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Amit Kumar
G. K. Goswami
Edwin Huffine Editors
Handbook
of DNA Forensic
Applications and
Interpretation
Handbook of DNA Forensic Applications
and Interpretation
Amit Kumar · G. K. Goswami · Edwin Huffine
Editors
Handbook of DNA Forensic

Applications
and Interpretation
Editors
Amit Kumar G. K. Goswami
BioAxis DNA Research Centre Private Ltd Uttar Pradesh Police
Hyderabad, Telangana, India Lucknow, India
National Law University, Delhi (NLUD)
Edwin Huffine
New Delhi, India
Huffine Global Solutions
Oklahoma, OK, USA National Forensic Sciences University
(NFSU)
Gandhi Nagar, India
Rastriya Raksha University (RSU)
Gandhi Nagar, India
Jindal Global law School
Sonipat, India
ISBN 978-981-19-0042-6 ISBN 978-981-19-0043-3 (eBook)

https://doi.org/10.1007/978-981-19-0043-3
© Springer Nature Singapore Pte Ltd. 2022

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
This book is dedicated to Padmashri Dr Lalji
Singh, The Father of Indian DNA
Fingerprinting.
Gandhinagar, India
Preface
DNA technology is a high end state of art technology in the field of molecular biology.
Its immense applications and authentic data generation capabilities have made it a
technology on demand by forensic and legal professionals. This book is not limited
to the boundaries of subject and domain as it is an application oriented science
with a plethora of implications. The book covers most of the important aspects of
DNA based technologies from the basic scratch to the sophisticated applicability in
the forensic area. Its contents can be used as a precious handout for Police, Judi-
ciary and legal practitioners and scholars who are interested towards the technical
aspects of DNA and its application in their domain of work. All the chapters are
presented by prominent individuals in this field. The book is a blend of information
related to DNA technology and its applications in the arena of Forensic investiga-
tion, Judiciary inspections and resolution of civil and criminal disputes. Being an
interdisciplinary handout, it is a great resource for individuals and authorities from
the Police department, Forensic institutions, Researchers and Medicolegal profes-
sionals. The information furnished in the book includes technical laboratory protocols
set along with chapters containing case studies. Role of DNA technology in disaster
victim identification is well elaborated in this book. Various technical concepts of
RFLP, STR Profiling, DNA Fingerprinting, LIMS, RFID, Biometrics etc, are well
explained. The authors of this book are practitioners and researchers from presti-
gious institutes which includes Indian Police Department, Internationally acclaimed
Forensic Professionals, Judiciary making the compilation a great knowledge archive.
Some of the chapters in the book are also focused on aspects and challenges faced
by technologies in Legislature and decision making. This can resolve the conflicts
related to ethical issues of DNA jurisprudence. Overall the compilation can influence
individuals from an array of sectors related to criminology and Forensic sciences.
This handbook can be advised and utilised for forensic and criminology researchers,
police officers, Doctors, lawyers and scholarly intellects.
Hyderabad, India
ix
Contents
Part I Insights and Advancements in DNA Forensics

1 DNA: The Master Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Amit Kumar, Amita Kashyap, and Edwin Huffine
2 Attaining State of the Art in DNA Tests . . . . . . . . . . . . . . . . . . . . . . . . . 11
Edwin Huffine, Amit Kumar, and Amita Kashyap
3 Inscription from Author’s Diary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Edwin Huffine
4 DNA Sample Collection and Packaging: Chain of Custody . . . . . . . . 35
Amit Kumar and Amita Kashyap
Part II Extraction and Analysis of DNA Samples in Laboratory

5 DNA Extraction from Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . 51
Amita Kashyap and G. Jyothsna
6 Post Extraction Processing of DNA for Profiling
and Understanding Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Part III DNA in Courtrooms

7 DNA in Criminal Adjudication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
G. K. Goswami and Siddhartha Goswami
8 Legal Contours for DNA Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
G. K. Goswami and Siddhartha Goswami
9 Forensic DNA in Exonerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
G. K. Goswami and Aditi Goswami
10 DNA Legislations, Challenges, and Opportunities . . . . . . . . . . . . . . . . 119
Lily Srivastava
xi
xii Contents
11 DNA-Based Human Identification in Mass-Disaster Cases . . . . . . . . . 149

Sachil Kumar
12 DNA as Tool for Revealing Truth in Civil as Well as Criminal
Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Gaurav and Sandhya Verma
Part I
Insights and Advancements in DNA
Forensics
Chapter 1
DNA: The Master Molecule
Amit Kumar, Amita Kashyap, and Edwin Huffine
Introduction of DNA
All living creatures have DNA (deoxyribonucleic acid) as their blueprint for life.
This DNA primarily exists in the famous double helix and is composed of four
bases, adenine (A), cytosine (C), guanine (G), and thymine (T), and these bases form
specific pairs with each other on the double helix (A with T, and G with C).
DNA or deoxyribonucleic acid is a genetic material present in the nucleus of cells
in all living organisms and DNA is unique for each and every individual just like
fingerprints. It is a DNA present in every one of us that makes us distinguishable
from each other. Hence DNA is an excellent identification parameter that differs from
individual to individual.
DNA analysis deals with DNA fingerprinting of all types of forensic samples of
human origin like blood, bloodstains, cement stains, vaginal swabs, tissues, bones,
teeth, and other skeletal remains.
Deoxyribonucleic acid or DNA is referred to as our genetic blueprint because
it stores the information necessary for passing down genetic attributes to future
generations. Residing in every cell of our body with the exception of red blood cells
which lack nuclei DNA provides a computer program that determines our physical
features and many other attributes. The complete set of instructions for making an
Organism the entire DNA in a cell is collectively referred to as its genome.
A. Kumar · A. Kashyap (B)

BioAxis DNA Research Centre (P) Ltd., Hyderabad, India
e-mail: amita@dnares.in
A. Kumar
e-mail: amit.kumar@dnares.in
E. Huffine
Huffine Global Solutions, Oklahoma, USA
e-mail: edhuffine@hotmail.com
© Springer Nature Singapore Pte Ltd. 2022 3

A. Kumar et al. (eds.), Handbook of DNA Forensic Applications and Interpretation,
https://doi.org/10.1007/978-981-19-0043-3_1
4 A. Kumar et al.
Half of the DNA is inherited from a person’s mother another half from his or
her father. Siblings inherit different combinations of DNA from the same parents
and are therefore different from each other. Each generation of people is a new and
different combination of genetic material from the previous generation. Except for
the identical twins each person’s DNA is unique, although the technology available
does not yet allow the examination of every single difference between people’s DNA.
In total, human DNA has approximately 3 billion bases. Long sequences of these
bases give rise to various genes and specific genes code for specific proteins by first
being transcribed into messenger RNA (mRNA) and then the mRNA being translated
into proteins. Most human DNA does not code for any specific protein and is often
referred to as “blank DNA”. Just as there is great variety among humans, so there is
great variety among the genes, and from this, there is also variety among the DNA
sequences of the A, C, G, and T that comprise the genes [1].
Human DNA is divided into 23 pairs of chromosomes, which contain the total
of the ~3 billion bases of DNA [2]. Genes that code for certain proteins are always
found in the same location on chromosomes among all people. Difference in base-
pair sequences within the genes gives rise to alleles, which are different varieties of
the same gene. In addition, groups of people whose ancestors originated from various
parts of the world have alleles that are found to have originated from that specific
location. This has given rise to several ancestry tests where individuals submit their
DNA samples to obtain an analysis of their ancestors. Even though the DNA between
humans is more than 99% similar, it is these differences that are targeted in forensic
investigations. With the exception of identical siblings, everyone’s DNA is unique
to that person, and there are indications of some variability between identical twins.
It is this uniqueness that gives DNA its power to identify.
In humans, there are two sources of DNA: mitochondrial DNA (mtDNA) and
nuclear DNA. MtDNA comes from organelles located in the cytoplasm called mito-
chondria, while nuclear DNA is located in the nucleus of the cell. The mtDNA is
much smaller than nuclear DNA and is generally around 16,570 base pairs in size.
This DNA codes for the replication and operation of the mitochondria, which are
responsible for the production of energy in the cells. Each cell may have dozens of
mitochondria and each mitochondrion may have multiple copies of its DNA, meaning
there could be hundreds of copies of mtDNA in a cell. MtDNA is maternally inher-
ited, meaning a mother and her children will have the same mtDNA sequence, with
the exception of when mutations occur. There are regions in the mtDNA known as
hypervariable regions, that do not code for proteins and thus, whose conversation
of their sequence is not critical to the functionality of the mitochondria. Therefore,
these areas are more prone to mutational events, which give rise to variability in the
mtDNA sequence. It is these differences that are used to link a sample to a maternal
lineage. As all maternal relatives should have the same mtDNA profile, mtDNA
does not point to identity but can be used to help support the linkage of a sample to
a particular individual. Also, as there are hundreds of mtDNA sequences per cell,
there are times mtDNA may still exist within a cell after nuclear DNA is no longer
obtained and testable [3].
1 DNA: The Master Molecule 5
Nuclear DNA exists as a pair of 23 chromosomes in our cells, with the exception
of gametes, which contain a single set of 23 chromosomes and the complete set of 23
chromosomes is obtained when an egg is fertilized. A person gets half of their nuclear
DNA from their father and half from their mother. In this way, the nuclear DNA in
humans constantly changes from one generation to the other. This much larger source
of DNA is responsible for the physical characteristics of individuals and is unique
to each person, with the exception of identical twins. Nuclear DNA is unique to
each individual and its profile points toward individual identity. As more is learned
regarding the frequencies of various sequences of DNA bases, an improved likelihood
of finding a particular sequence is determined. As dozens to hundreds of locations on
nuclear DNA are often tested and their sequence determined, a statistical statement
of the odds of someone else who is not the suspect or missing person presenting the
same DNA profile can be created.
The Master Molecule in Witness Box (with Some Case

Studies)
The advent and continual evolution of the science and capabilities of forensic DNA
testing directly impact the ability to identify and reassociate individuals who were
killed in mass loss of life incidents, as well as the ability to identify individuals in
single graves. This ability began in the 1980s with the first application of forensic
DNA testing in a courtroom setting. At that time, forensic DNA testing was a rela-
tively new science and many of its principles and intricacies were not well understood.
As it was a new science that was not well understood, it was required to undergo and
successfully navigate a series of in-depth validation studies. The series of valida-
tion studies that forensic DNA testing was required to undergo prior to widespread
acceptance created a standardized system that was acceptable around the world and
that was far more objective in nature than many other forensic sciences. As the odds
of a DNA match between a recovered and tested sample and some type of reference
sample are dependent upon the frequency of alleles, bases, or markers, and as the
frequency of these alleles occurring within a population is known from previous
testing, a statistical probability of a match can be determined. Being able to assign
such statistical modeling combined with the testing being objective and reproducible
are several of the key reasons forensic DNA testing has often been called the “Gold
Standard” of forensic testing [4].
DNA testing supports the rule of law by not only matching unknown samples to
known individuals, but also by exonerating those who are charged or suspected of
crimes and in which the DNA testing reveals it is not their sample left at a crime scene.
In this way, DNA testing can remove suspects from suspicion, as well as link samples
from crime scenes to individuals as well as to samples from other crimes scenes, thus
aiding in the understanding of who may have committed criminal activities as well
as the relatedness of various crimes. The more robust a sample collection and DNA
6 A. Kumar et al.
testing system is, often times the greater its impact on society by solving crimes,
removing criminals from the streets, and lowering overall crime rates. In addition,
testing of samples from past cases helps to exonerate those who may have been
incorrectly convicted of crimes they did not commit and this has led to several
Innocence Projects in which cases are reviewed to determine the availability of a
biological sample and the circumstances surrounding the conviction and if doubts
about the conviction are created in the reviewers. The testing of samples in this
manner has led to hundreds of people being exonerated from past convictions. By
reducing crimes rates, which results in fewer victims, and releasing suspects from
incorrect suspicion of committing crimes, the state is held more accountable to the
people, thus supporting human rights.
One example of how a robust DNA testing project can reduce burglary was
performed by the FBI in Denver, Colorado. The focus of this project was to collect
and quickly test biological samples that were collected from crime scenes related
to burglary. Samples collected were compared to various suspect DNA databases
as well as to DNA profiles obtained from other burglary crime scenes collected in
Denver. The results of this project demonstrated how linking DNA profiles obtained
from crime scene samples across a city can develop a better understanding of the
individuals involved in such crimes and aid in investigations. Once one suspect is
taken into custody and their DNA profile linked to a series of crimes, knowing
with whom they associate may lead to additional arrests. In the case of the Denver
project, the crime rate for burglary fell significantly and the consequential decrease
cost to the police, courts, and property owners more than outweighed the cost of
the DNA testing program, meaning the implementation of a robust DNA testing
system to combat burglary resulted in a net financial savings to the city, as well as an
incalculable mental benefit to homeowners who did not become victims of burglary
[5].
As our understanding of the power of DNA testing has increased and improved,
how it can be better utilized in legislation has changed. A DNA profile from a
crime scene remains the same with the passage of time, unlike the memory of a
victim, which may be inaccurate and change with time. Strong emotions may also
suppress memories, or alter them to the point that would lead to false convictions.
Various legislative changes incorporated the unchanging nature of DNA to develop
relatively unique strategies to more fully gain the benefits of forensic DNA testing.
For example, the testing of rape kits can often provide a DNA profile from the rapist,
even if the rape occurred decades ago. Some states permit the charging of a DNA
profile obtained from a crime scene with criminal counts. This effectively froze time
passing in the statute of limitations and the person can later be arrested and charged
with the crime once their profile is obtained [6]. Another lesson learned is the value
of creating local DNA database, in addition to a national database. Most crimes are
committed by people who live in, or nearby, the city where the crime occurred, thus
providing greater value to a local database. Also, local authorities can more often
than not prioritize cases and obtain results in a shorter amount of time, which results
in expediting justice.
DNA Forensic Investigation
Of all the functions of the police, investigation is the most important and vital one. In
the constantly evolving socio-economic scenario, the criminals using sophisticated
tools and techniques commit more and more crimes. In order to overcome these
complexities, the police all over the world are depending more and more on scientific
methods of investigation. A wide range of scientific techniques is now available for
analysis of varied nature of objects and materials encountered in the process of
Commission of crime by the culprit in and around the crime scene, on the suspect
and victim. The study of such material evidence also known as objective evidence or
physical evidence applying the latest scientific tools and techniques for proving the
guilt or innocence of the accused by the courts of law is broadly known as forensic
science.
The application or aid of science to the crime investigation is fundamentally
one of reconstruction, that is, trying to assist in determining what happened, where
it happened, when it happened, and who was involved. It is not concerned with,
and cannot determine, why something happened. Forensic analysis is performed
on evidence to assist the police and the court in establishing physical facts so that
criminal or civil disputes can be resolved. It is the job of the forensic scientist to
translate the legal inquiry into appropriate scientific questions and to advise the
investigating officer and or the judiciary on the capabilities, limitations, and results
of the analytical techniques.
In forensic science, the laws of various facets of science are applied in conducting
an analysis to determine the nature and characteristics of physical evidence collected
in the process of crime or civil investigation. Using the scientific methods, inferences
are drawn about how the evidence can build into the crime and criminal. These
inferences are thus connected to the events that may or may not have taken place
in connection with said evidence. The law defines elements of a crime; science
contributes information to assist in determining whether an element is present or
absent.
It is an established fact that the criminals while committing crimes either due
to carelessness or due to anxiety, or due to contact with other objects leave traces
at the scenes and these are the basis for scientifically exploiting their culpability.
This physical evidence when located, collected, preserved, and forwarded for the
scientific evaluation, the report is bound to have in rumors potential in linking the
criminal to the crime scene, victim, or any other circumstances leading to the crime.
Moreover, evidence collected and based on scientific evolution and report is unbiased,
universally acceptable, and also stands the test of time.
There is a time limit for sending physical evidence to forensic laboratories for
scientific analysis. The investigating officer should collect and forward all types of
physical evidence along with controlled samples so as to reach the forensic labora-
tories as soon as possible from the date of crime. Physical evidence sent after around
30 days should not be accepted except in rare cases that too with a proper justification.
8 A. Kumar et al.
The success of the scientific effort in arriving at the truth depends both on the role
of the investigating officer and the forensic scientists that are in the thorough search
and collection of evidence in the proper way by the investigating officer and then
careful analysis through the right tools and techniques by the scientists.
Purpose of physical evidence in crime investigation: The world physical
evidence denotes all evidence having certain physical dimensions such as size, save,
pattern commands length width height, volume, etc. which can be preserved, lifted,
collected, packed, and forwarded for the scientific analysis to the space list concern.
Physical evidence can alternatively be called as material evidence, objective evidence,
circumstantial evidence, or indirect evidence in different contexts. Physical evidence
includes all naturally occurring substances as well as machine or man-made objects
in the universe.
Goal of Physical Evidence: The goal of physical evidence examination is to
provide useful information for the criminal investigators in solving crimes and for
courts of law during the education of these cases. The following leads can be obtained
from the examination of physical evidence in the process of investigation.
The corpus Delicti: The corpus delicti refers to those essential facts which show
that a crime has taken place. For example com tool marks, broken doors or windows,
ransacked rooms, and missing valuables would be important in establishing the
burglary has taken place. Similarly, in an assault case, the victim’s blood or torn
clothing could be important pieces of physical evidence.
Linkage: The linkage is one of the most common and an important aspect that
physical evidence can help to establish. Blood, hairs, clothing fibers, cosmetics, and
other items from the victim may be transferred to a perpetrator. Items found in a
suspect’s position can sometimes be linked to the victim, for example through the
comparison of bullets with a weapon seized from the suspect the suspect can be
linked with crime. It is also possible that evidence is transferred from a perpetrator
to the victim in rape, murder, and assault cases.
Linking a person to a crime scene: It is one of the most vital steps in a crime inves-
tigation. Numerous types of evidence may be deposited by the person committing
a crime, including fingerprints, footprints, blood, hair, fibers, and soil. In addition,
the type of weapons or objects used may also leave evidence for example bullets
and cartridge cases or tool marks. Depending on the type of crime, various kinds
of evidence from the scene may be carried away intentionally as a story property
and unintentionally as a transfer of tracks evidence such as carpet fibers or hairs on
the criminal’s shoes or clothing. These materials are extremely useful in linking an
individual to a particular crime scene.
Disproving or supporting a witness’s testimony: Physical evidence analysis can
often indicate conclusively whether a person’s version of a set of events is credible,
or whether an alley put forth is convincing or not. For example, the examination
of a car that fits the description of a hit-and-run vehicle might reveal blood on the
underside of the bumper. If the owner of the vehicle claims he hit a dog, laboratory
tests on the blood can reveal whether the blood is from a dog or from a human and
further testing for DNA profiling may reveal the truth relating to the accident and
victim.
Identification of a suspect: The best evidence for identifying the suspect is his or
her palm prints and evidence such as blood, semen, saliva, skin tissues, etc. collected
for DNA fingerprinting examination. A fingerprint found at the scene, and later
identified as belonging to a particular person results in an unequivocal identification
of that person has having been at the scene. The term identification when applied to
people really means individualization.
Providing investigative leads: Physical evidence analysis can assist the investi-
gator in pursuing a productive path, by providing close from the characteristics of the
physical evidence. In a hit-and-run case, for example, examination of a chip of paint
found in victim’s clothing could be used to provide information on the color and
possibly the model and year, of the automobile involved. With the rapid emergence
of computers add fingerprint and DNA databases, the ability of physical evidence to
provide investigation leads has increased enormously.
Crime scene reconstruction: Transient evidence such as odor, temperature,
imprints, marks stains-pattern evidence like bloodstain patterns, skid mark patterns,
guns out at residual patterns, projective trajectory patterns, glass fracture patterns,
etc. and conditional evidence such as light, smoke, fire, what are, piled up newspa-
pers, etc. will play a key role in reconstruction of the events surrounding the crime
scene and the nature of office. Sometimes it may also give valuable leads regarding
the perpetrator based on the individual traits in relation to the evidentiary pattern.
Developing DNA Databases and Revolutionizing

the Forensic Investigation
The basic premise of using DNA profiles to combat crime and/or identify the missing
from unknown samples is based upon putting the DNA profiles obtained from samples
at crime scenes into one database and comparing to DNA profiles obtained from
suspects that are stored in another database, or from DNA profiles obtained from
skeletal remains and comparing them to DNA profiles of family members who are
missing family members, or personal articles of the missing that might contain some
of their DNA. The ability of DNA testing to match profiles obtained from unknown
samples to a known reference grows as the size of the DNA databases grows. For
criminal investigations, from whom samples can be collected for entering into the
“known” or reference database, is a primary determinate on creating a successful
DNA matching project. When databases were first being created, most legislation
required samples only be collected from convicted felons and even then, for only
the most serious crimes such as murder, rape, and crimes against children. As many
of these felons were in prison for an extended period, this meant that testing of
samples from crime scenes had a low chance of resulting in a match. As the power
of DNA database became better understood, legislation changed to collect samples
from a wider population [7]. The types of crimes from which suspects/convicted
would be collected grew and with each increase in collection categories, the rate
10 A. Kumar et al.
of matching crime scene samples against known samples increased. Some nations
and states included collecting samples for lesser crimes, such as traffic offenses,
which dramatically increased the power of DNA database. Some nations and states
in the United States passed all arrestee legislation, in which samples were collected
from individuals who were arrested of various crimes. If the individual was later
exonerated and/or the case dismissed, their samples were to be expunged from the
database of known samples, though in some cases this could be a lengthy process.
While some nations are considering total population DNA database where everyone
is required to provide a sample, such efforts have been strongly resisted based upon
concerns for civil rights and liberties.
As stated above, the power of a DNA database to link DNA profiles obtained
from unknown samples to know samples is directly related to the number of samples
contained within the database. The UK undertook an aggressive collection effort
in which samples were collected from individuals involved in low-level crimes, as
well as those convicted of serious crimes. This encompassing database resulted in
the UK system having a match rate of an unknown sample to a known person of
approximately 60% [8]. In the context of Bosnia and missing persons due to conflict
from the breakup of the former Yugoslavia, as the database of family members who
are missing family members grew, the chance of a set of unknown skeletal remains
matching family references exceeded 80%. The number, and from whom, samples
are collected is a prime factor that will determine the power of a DNA database.
References
1. https://medlineplus.gov/genetics/understanding/basics/dna/.
2. https://www.genome.gov/about-genomics/fact-sheets/Chromosomes-Fact-Sheet
3. https://medlineplus.gov/genetics/chromosome/mitochondrial-dna/.
4. https://www.dw.com/en/dna-fingerprinting-the-gold-standard-of-all-identification-methods/a-
36045424.
5. https://www.youtube.com/watch?v=M1q4gEMKve4.
6. https://www.mcclatchydc.com/news/crime/article24573589.html.
7. https://scholarlycommons.law.case.edu/cgi/viewcontent.cgi?article=1545&context=health
matrix.
8. https://www.sciencedirect.com/science/article/pii/S2589871X19300713.
Chapter 2
Attaining State of the Art in DNA Tests
Edwin Huffine, Amit Kumar, and Amita Kashyap
In the early days of DNA testing, the most sensitive type of testing was via a process
known as Restriction Fragment Length Polymorphism testing, or RFLP. In this type of
testing, DNA was partially digested, or cut into pieces, by using a variety of enzymes
that cleaves DNA at a certain runs base pair sequences [1]. The resulting fragments
were then tagged with radioactive P32, run on a gel by electrophoresis, and the gel
was then placed on photographic film. The P32 would arrive at the laboratory in a
lead ‘egg’ that blocked the radioactivity. Laboratories needed rigorous protocols for
handling radioactivity and on more than one occasion, the accidental transfer of P32
radioactive outside of its containment area would shut down laboratory operations for
a time. After several days, the film was developed and the RFLPs that contained the
P32 showed up on the developed film as bright ‘spots.’ These spots were measured
and the length of which they traveled on the gel was compared to known sized samples
that were run as controls. The spots were then placed into ‘bins,’ or a range of size
standards, into which they fit based upon migration distance. This was a laborious
process that required what by today’s standard would be an enormous amount of
DNA and that relied on using radioactivity and a significant amount of time. The
bins in which the various RFLPs were placed was not always a simple and straight
forward process to determine and some variability in how the bins were called could
result, even among scientists reviewing the same developed film. However, the use
of radioactivity was one of the more sensitive ways to detect DNA prior to the
introduction of the PCR process.
E. Huffine (B)
Huffine Global Solutions, Lawton, USA
A. Kumar · A. Kashyap
A. Kashyap

https://doi.org/10.1007/978-981-19-0043-3_2
12 E. Huffine et al.
The introduction of the PCR process removed the need to rely on radioactivity and
created a wide range of new DNA testing options. Some of the first new applications
in the early 90s involved the amplification of certain known and categorized segments
of that displayed variability among people. The amplified products were then ‘bathed’
on strips of specially treated paper in which some regions had complimentary probes
attached. After this bathing process, the strips were developed and the particular spots
that changed color revealed the particular sequence for this locus. This methodology
made the calling of a genotype more standardize and, by application of the PCR
process, permitted the testing and profiling of far smaller quantities of DNA and
permitted a significant expansion in the application of DNA testing.
The next great leap in DNA testing involved capillary electrophoresis in which
amplified DNA fragments were tagged with fluorescent probes during the PCR
process [2]. In this methodology, the fragments separate out based upon their size,
with the smaller segments traveling faster through the gel. A particular ‘color’ of the
probe corresponded to a particular base in the DNA. The amplified and tagged DNA
profiles were run on a slab gel, or through a column during which time they were
exposed to various wavelengths of light, resulting in the fragment fluorescing and
being detected.
As the science and understanding of DNA testing became better understood, how
it could be applied in additional ways began to be realized. In addition, the sensi-
tivity of the instrumentation increased and newer STR tests kits provided increased
abilities to obtain DNA profiles from degraded samples. The ability to obtain DNA
profiles from samples containing ever decreasing quantities of DNA and also from
degraded samples dramatically increased the type of samples and cases in which
DNA profiles could be obtained. One area in which these improved technologies
were applied was for testing of backlogged samples from rape kits as well as other
criminal and missing persons’ cases. Automation of the extraction, amplification,
and DNA profiling process increased the speed with which DNA profiles could be
obtained, many laboratories are now involved in reducing the backlog of cases, some
of which have been in storage for decades. The mass testing of backlogged rape
kits has produced DNA profiles that belong to the alleged attacker and has revealed
instances of serial rapists. Even if, in some cases, the individual cannot be charged
due to the statute of limitations, it does still provide information on who was involved
in these crimes. One example of many includes testing of samples related to episodes
of violence and civil unrest and how DNA testing may be able to help deter future
violence by demonstrating the ability to hold individuals accountable for their actions.
This type of testing can be used to gain a better insight on who may be involved in
campaign of mass rape that is used as a way to intimidate a population. If samples
are collected from the victims of rape and tested, this testing will show if the rape
kits have semen from multiple men. By testing a large number of samples, it would
be possible to link Y-STR profiles from victims from multiple locations together to
help in understanding if a particular group of men were involved in multiple rapes
across a wide range of cities. If such a group is able to travel in an area of unrest, a
question of if they were supported arises. It should be noted that Y-STR profiles are
paternally inherited, meaning more than one male will have the same Y-STR profile
2 Attaining State of the Art in DNA Tests 13
and this needs to be taken into account. Once additional evidence associated with a
series of mass violence incidents is obtained and combined with witness testimony
and DNA profiles, understanding why certain patterns exist in which DNA profiles
are found among the various locations can be better understood and this information
can be submitted to the legal-judicial systems and courts.
It is this ability to obtain DNA profiles from samples collected from even decades
ago that provides investigators with additional options for closing cold cases, or
better understanding those involved in crimes. DNA testing on biological samples
from past crimes can be used to support, or refute, the convictions of individuals
whose conviction was made before the regular use of DNA testing. This has resulted
in the creation of many ‘Innocence Projects’ around the world, and those who were
wrongly convicted are being released once the DNA evidence shows that is not their
biological sample that was tested. In this way, the stated is held more accountable to
their citizens and is a great support of human rights.
In addition to the increasing ability to obtain DNA profiles from biological
samples, there is also an increasing knowledge of which genes produce physical
characteristics, such as hair color, eye color, etc. [3]. Understanding the specific DNA
sequences that produce these traits allows for some type of phenotypic determina-
tion of the person who left the biological sample. This is still an area of significant
research and is complicated as often times a series of different genes are responsible
for physical characteristics. As our understanding on which genes are responsible
for which physical characteristics increases, the ability to perform genetic testing to
reveal the physical traits of the person from whom the biological sample originated
will improve. Another complicating factor to use genetics to determine the physical
appearance of an individual is the impact environmental factors have on how fully
genes will manifest their characteristics. For example, the genes of may indicate he
could reach a height of 6 2 –6 5 , but if he has insufficient nutrition during part of
his maturing years, he may not reach that height range.
Among the newer forms of DNA testing is a method called next generation
sequencing. This is a technique that can sequence the entire human genome and
does so by amplifying and sequencing various sequences many times over and then
a composite DNA profile is developed by computer programs that line up and the
amplified and sequenced fragments and calling the base at each location [4]. This
type of DNA testing provides the maximum amount of data possible as far as the
DNA sequence of an individual and may be able to discern between identical twins
as some variability exists between identical siblings. Another way to increase the
amount of DNA data obtained from a sample is to use single nucleotide polymor-
phisms (SNPs). This technique determines the base at a particular location on the
DNA and thousands to millions of loci can be tested at the same time. While any
one SNP loci may not have a significant ability to aid in identification, when multi-
plied by thousands or millions of sites, this technique becomes incredibly powerful.
This type of testing is the foundation of many ancestry testing companies in which
a person submits a sample of their DNA and get results back detailing their distant
lineages based upon a region/s around the globe [5]. As databases continue to grow
around the world, this type of ability to show ancestry will become more refined
and precise. A benefit of using SNPs and/or whole genome sequencing in human
identification is the ability to go to more distant relatives for reference samples. In the
standard STR kits that have 16–20 loci that are often used, having relatively close
relatives, such as parents, children, siblings, and nieces/nephews are most benefi-
cial. This somewhat limits the effectiveness of STR testing when these reference
samples are not available, which can occur for missing persons from conflicts of the
more distant pass, like the missing from World War I. When DNA replicates, it is
somewhat ‘sticky’ and pieces of DNA from one chromosome can become added to
another chromosome, meaning thousands to millions of bases in a length of DNA
could become attached to another chromosome and when this happens to a gamete
cell, the resulting DNA becomes incorporated into all the cells of the newly formed
embryo and thus, the child. This leads to the ability to use more distant relatives,
in some cases up to 3rd cousins, as references as even these distant relatives may
share thousands to millions of bases in a sequence of DNA. An interesting point for
consideration is that, given that 3rd cousins may become suitable references when
SNP and whole genome sequencing is used, the majority of the US population is
covered out to 3rd cousins in the ancestry databases. This has the implication that
the majority of missing persons in the US could be identified by using this technique
and comparing the profiles from the missing to those in these databases.
Studies and research are underway in which damaged DNA can be repaired.
Though this technique still has a way to go before being used in the forensic setting,
it offers the hope of being able to repair damaged DNA and thus, to open new avenues
in obtaining DNA profiles from even highly damaged DNA. If this technique proves
to be conservative, meaning that the same DNA bases are inserted in place of damaged
bases, this would open a world of possibilities in the world of using DNA testing to
identify the missing.
Forensic DNA testing for the identification of the missing and to solve crimes
has rapidly evolved over the past 35 years. The science of forensic DNA testing has
evolved, as has our understanding of the how it can be applied to an ever-increasing
variety of samples. The legal-judicial/courtroom setting has also adjusted to this new
forensic technology and in doing so, not only holds individuals accountable to the
state, but also the state accountable to its citizens. By returning the names of the
missing to their remains and aiding in solving crimes and exonerating the innocent,
forensic DNA testing is a great support of human rights. This technology continues
to improve in both capabilities and decrease costs and time of testing, as well as it
becomes mobile in the form of mobile DNA laboratories that can be transported to
where needed, and will be an increasing part of our world in the future.
Forensic Bioinformatics
Bioinformatics is the application of information technology in the storage, retrieval

and analysis of biological information. This application would enhance the rate of
research and generation of new data. In Bioinformatics the three main concepts
include storage of the data in properly structured and formatted databases, a proper
retrieval system for collection of stored data and the generation of new data through
analysis. This requires investigation of stored data using various algorithms and
statistical techniques. All these concepts of Bioinformatics when applied to genetic
analysis has laid the foundation for a new domain of science termed Forensic
Bioinformatics.
Forensic Bioinformatics is the interdisciplinary domain of science dealing with
the application of biological principles in forensic investigation to resolve judiciary
cases. Bioinformatics and Forensic science draw their techniques from computer
science and statistics with the aim to resolve the judiciary problems using biological
knowledge [6]. Forensic Bioinformatics enables the establishment of DNA databases
including the STR profiles of all the known offenders and persons identified in several
crime investigations. The current advancements in Bioinformatics and development
of MPS massively parallel sequencing for analysis of DNA enabled the identification
of vital facts and resolving sensitive issues. Forensic Bioinformatics has become an
indispensable weapon in forensic crime investigations. Forensic Bioinformatics runs
on the pillars like open access forensic DNA databases including ExAC, genome,
exome aggregation database etc.
Use of Forensic Bioinformatics in handling mixed DNA samples and resolving
unsolved cases: Crime scene evidences inculcating mixed DNA profiles in analysis
has always been a problem in crime investigation. In addition to introduction of
confusion in the case it may lead to misguidance and fatal errors in investigation
making it a vital concept to be resolved [7]. There emerged Forensic Bioinformatics
techniques for analyzing the STR data of the samples that can yield high end accuracy
and unbiased results for investigation. The analysis of Short Tandem Repeats for
the evaluation of specific loci within nuclear DNA utilizes an ‘MS-Excel database
management tool’ for concluding the case. In this Bioinformatics based technology,
management and analysis of DNA profiles obtained from Forensic laboratory is
performed based on various loci present and their individual specific variability.
This allows the resolution of unsolved cases in Forensic science.
LIMS
Laboratory Information Management System is a software platform enabling effi-

cient storage of data related to samples and corresponding annotations. It is also
referred as LIS or LMS which means the same. LIMS is a boon for automated work-
flow, instrument integration and sample and information management [8]. This soft-
ware provides an advanced and efficient online laboratory management and research
with good accuracy and authenticity. There is a drastic evolution of LIMS from
a basic sample tracking system to an enterprise resource planning tool managing
various aspects of laboratory informatics. It encompasses vast domains of laboratory
informatics.
All LIMSs include a workflow component and a facility for summary data manage-
ment. However there are significant differences in the functionality among each other.
From the time of its establishment LIMS has transformed to a vast extent and in
current trend it is also applicable in data mining, data analysis, maintaining electronic
laboratory notebook etc. Further integration is also added to several LIMS systems
which enable study and analysis of translational medicine on a single stationary
platform.
LIMS in Forensics: LIMS in forensics has become a vital tool for forensic labo-
ratory, streamlining the documentation, evidence management, sample accession,
extraction, quantification and analysis. This system can provide out of the box func-
tionality which can be configured to meet the needs of the laboratory. LIMS enables
data input, sample documentation and quality control. The replacement of regular
laboratory data maintenance with LIMS provides results at a faster rate with high
accuracy and compliance [9].
In Forensics LIMS manages the complete exercise from pre logging of evidence,
crime scene documentation, lab analysis, storage of property and its disposition etc. It
perfectly streamlines all the forensic processes with reduced time consumption, cost
and achieves compliance. It makes the collection of evidence easy and accessible.
This system drastically cuts down the paper work and enables to have a strong focus
on every single aspect of the case.
LIMS in DNA data maintenance: This system retrieves the sample information
and generates the worksheets required for all analysis processes in DNA including
sample preparation, DNA extraction, quantification of DNA, amplification and anal-
ysis. Worksheets are designed with required columns for the input of on-going work-
flow information and also the option to print the page if necessary. The user can start
interacting with the software after logging in with the credentials provided like user
id and password which enables restrictions in the users and retains data confiden-
tiality. There is a strict control over the data options that can be edited with a strong
audit trail that can track the changes. Exchange of the LIMS data with multiple DNA
instruments is one of the added features. A load list is produced for the instrument
either through network connection or manual transfer. The results file should be
imported to the LIMS (Fig. 2.1).
The LIMS as shown above includes vital component as sample management
of forensic investigation to the simplest component of quotation and invoicing
eliminating the human interruption and thereby maintaining compliance in data
management and accuracy.
RFID
Radio Frequency Identification system (RFID) is a means of tracking and identifi-

cation system employed by Law enforcement agencies and Forensic experts [10].
This is applied for sample tracking and management of evidences, individuals and
firearms. This is one of the automated identification technology (AIT) streamlining
Fig. 2.1 Pictographic representation of various components in LIMS system. Picture Courtesy
Autoscribe informatics. https://www.autoscribeinformatics.com/resources/blog/what-is-a-lims
the steps of capturing, collecting and transferring of the data for the identification and
tracking of individuals and assets. This technology provides a high end applicability
and reliability in accurate location identification, measurement of environments and
automatic updation in the change of position and condition of the individuals or
properties.
Working principle of RFID: It is completely based on the radio frequency signals
which consists of a RFID reader and a tag for the identification and tracking of the
personnel’s or objects [11]. This is advancement in the barcoding technology where
each object is identified by a unique reader. RFID system allows the identification
of an object falling within its proximity range. These RFID tags can be scanned and
read without the human intervention making the work much simpler and faster. These
RFID systems are more commonly employed in supermarkets, hospitals, hotels etc.
wherein they are applied for billing, data locking and security, door locks, attendance
systems etc. (Fig. 2.2).
The above block diagram represents various components in RFID along with the
direction of flow of signal toward the microcontroller. The two main components
in RFID include a microchip and an antenna. Microchip is a semiconductor device
sourced with data storage capability of some KB which can be transmitted upon
requirement. The antenna receives the stored data from microchip and transfers to
the reader for detection.
RFID technology in Forensic investigations: RFID systems perform automated
data acquisition with high precision and low time. Three types of RFID system are
known which include active, passive and BAP battery assisted passive RFID. Major
applications of RFID in investigations include:
(1) Automates the real-time tracking and tracking of the data and objects
(2) Expedites the processing of evidence for law enforcement agencies.
(3) Exhibits its support to government and investigation system in maintaining
evidence compliance standards
Fig. 2.2 Basic construction and working principle of RFID system. Picture Courtesy compo-
nents101. https://components101.com/articles/introduction-rfid-modules-construction-types-and-
working
(4) All the crime scene samples and evidences are monitored and safe guarded
during the entire protocol from sample collection to case resolution.
(5) Restricts the evidence contamination or intentional tampering by the individ-
uals thereby maintaining the fidelity of the case.
Apart from the LIMS and RFID systems there are several other investigatory and
sample tracking systems that enable the modernization of forensic investigations.
These technologies include Biometrics, Artificial intelligence (AI) and Internet of
Things (IoT).
Biometrics
This technology relies on identification of an individual or object based on measure-

ments and calculations preloaded in the software. These technologies are commonly
applied in face recognition, fingerprint identification, iris scanning etc. This tech-
nology has merged into several public sectors like colleges and organizations for
attendance, markets for product identification and billing, object identification etc.
The more advanced and modern application of Biometrics is in DNA Technology
[12].
DNA biometric identification of an individual is undefeatable and most accurate
in identification of an individual which is digital and will not change for a person
in his lifetime. It remains constant throughout assisting reliable personal identifica-
tion. STRs act as unique identifiers for generating biometric code. These codes are
completely based on STR short tandem repeats. These are unique for each individual
and responsible for generating a unique profile pattern. Another identification pattern
for uniqueness of an individual is SNPs single nucleotide polymorphisms yielding a

different genetic makeup.
Both STRs and SNPs can provide most reliable data for unique identification
and generation of DNA biometrics. Major limitations for this technology include
ethical issues and non-discrepancy of these profiles in monozygotic twins. Biometric
based individual identification plays a key role in forensic investigations by means
of databases and data analysis.
AI and IoT in Forensic Investigations
Artificial intelligence is the current day smart technology which deals with the
machines that behave as human brain and function independently. It is a broad area
of computer science involved in building machines which can potentially work effi-
ciently in managing tasks involving human intelligence. One of the basic examples
corresponding to AI is robotics. This is an interdisciplinary area with a vast horizon
of applications. Advancement in the AI technology is creating a paradigm shift in
various sectors forensic science being one among them [13].
AI can be simply defined as the technology involving machines with following
work characteristics:
1. Thinking humanly
2. Thinking rationally
3. Acting humanly
4. Acting rationally.
AI in Forensic Science: Due to the advancements in automated forensic data
generation and storage there is an urge in the need for tools in analyzing large data
sets which is fulfilled by AI technology. AI can be employed in metadata analysis
and pooling of the data for simplification and sorting within a short span of time.
Pattern recognition forms one of the principle techniques in Forensic science
which involves identification of different parts of an image or a person. Other cate-
gories of image recognition include identification of patterns either in emails and
messages or in sound files.
Pattern recognition relays on statistical methodologies and probabilistic
reasoning. AI system aids in recognition of such patterns with high accuracy and
authenticity. AI system treats the forensic samples to generate legal conclusions
with high end intellectual processing.
Another feature of AI aids in proper communication between different individ-
uals in forensic investigation like doctors, forensic statisticians, lawyers, criminal
investigators etc.
Another important applications of AI system includes repository creation,
building of statistical evidence, knowledge discovery etc.
IoT in Forensics: The term IoT refers to all the billions of instruments which are
working in connection with the Internet and can share their information globally.
This includes all systems that are inter connected and communicated with Internet
that can enable data transfer over a wireless network. IoT Forensics is a branch of
digital forensics which includes cases like IoT related cybercrimes which requires
investigation of the connected devices, sensors and all the devices involved in storing
the data. An increasing number of IoT devices in daily life like smartphones, laptop
etc., increase the chances of leaving forensic traces in crime investigations. This
technology also enables to recognize, preserve and prioritize the sample traces in
crime scene [14].
Technical Challenges to DNA Profiling and Forensic Analysis
Challenging, Degraded and Unusual Samples
In spite of the prominent and advanced reports generated by DNA testing in Forensic
investigations there are a bundle of challenges to this technology. Most sensitive
concept of this technology lies in DNA extraction from crime scene sample [15].
Some of the commonly faced challenges include insufficient sample for DNA extrac-
tion, mixed and cross contaminated samples, decaying and decomposed sample etc.
which requires an intellectual trouble shooting that should not hinder with the authen-
ticity of result. Some of the alternative techniques for such challenging cases are
specified here under.
Touch DNA/Trace DNA
As the term denotes, this technology is based on the principle that DNA fingerprint
can be obtained from even the actual fingerprints of an individual in critical cases.
This touch DNA technology is opted in the situation where in the sample quantity
obtained from the crime scene is below the recommended threshold in any case of
DNA analysis. Though this technology was reported long back by Van Oorschot and
Jones in 1997 it is practically employed in recent times. This concept of obtaining
DNA profiling from touch DNA fingerprint increased the number of samples to be
investigated and collected from crime scene location thereby increasing the chances
of case resolution in several unresolved cases [16]. This enormously increased the
number and types of cases that can be resolved using DNA technology. Though this
technology is accurate and cost effective there exists certain limitations which include
collection of mixed samples providing unreliable profiles, coincidental matches or
matches in profiles for identical twins called Chimeras limiting the applicability of
this technology.
DNA from Harsh Environmental Samples
Another challenge with DNA profiling for the crime scene samples is the degraded
sample or samples from harsh and damaging environmental conditions. Most of the
cases the investigation may restart a decade or more after the crime. This situation
poses a great challenge for DNA extraction and its implication in Forensic inves-
tigation. Environmental conditions which include humidity, temperature, bacterial
contamination, environmental moisture, dampness, UV irradiation are all factors of
the environment that challenge the integrity and quality of DNA obtained from crime
scene samples.
Obtaining DNA from Challenging Sources
As specified there are quite a lot of conditions in which the sample obtained is either
sufficient or not is a condition to yield best results using standard protocols. In such
cases use of robust techniques becomes a choice of the hour. Some of the solutions
at various steps of DNA analysis for resolving the problem are specified below [17]:
(1) Improving DNA Collection
• Use of novel collection device for increasing low copy number DNA
recovery
(2) Improving DNA quality and PCR efficacy
• Use of advanced high flow column for the extraction of DNA from
challenged bone samples
• Reducing the effect of inhibitors using PCT pressure cycling technology
• SCODA (Synchronous Coefficient of Drag Alteration) technique for the
efficient removal of copurified inhibitors from challenging samples
(3) Improving PCR of LCN (Low Copy Number) samples
Some of the techniques based on above specified principles include Pre
amplification, Mega Plex STR analysis, NGS analysis etc.
Pre Amplification: This technology involves robust extraction of DNA removing
inhibitors and enhancing product yield. This technology can also be employed for
challenging samples apart from the regular samples. Various molecular biology labo-
ratories supply advanced kits for the maximal extraction yield and quantification
decreasing the time consumed and increasing the chances of resolving the case [18]
(Fig. 2.3).
Mega Plex STR Analysis: One of the gold standard techniques for DNA extrac-
tion and yield enhancement is the use of STR profiling on capillary electrophoresis.
This can be used to resolve several forensic cases including missing person identi-
fication, burglary identification etc. Mixed DNA samples can also be used for this
kind of amplification and profiling.
Fig. 2.3 Steps in pre amplification
NGS technology for analyzing mixed DNA samples: The most advanced and
sophisticated technique of the generation is the Next Generation DNA Sequencing or
simply denoted as NGS. This technology involves the analysis of targeted and specific
forensic markers to generate profiles that can not only identify the individuals but
also separate the profiles from mixed samples. In cases of degraded DNA samples
in which traditional capillary electrophoresis may not yield complete profiles NGS
is the target of choice [19].
Figure 2.4 details the various steps involved in NGS sequencing. The steps include
(1) preparation of DNA libraries by fragmenting the DNA samples (2) enrichment
of Shotgun libraries with a DNA probe based method of targeted capture enrichment
(3) sequencing of enriched samples on NGS platform (4) analysis of nuclear SNP
data [20].
Fig. 2.4 Steps in next generation sequencing. Picture Courtesy Creative Commons Attribution 4.0
International
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Chapter 3
Inscription from Author’s Diary
Edwin Huffine
Just as the role of DNA testing in the legal-judicial setting has changed over time,
so has its role in the identification of missing persons. The remainder of this chapter
is based upon my personal experiences and work from over 30 years, primarily in
the missing persons’ field. This history begins close to the nexus of using DNA to
identify the missing, is involved in some of the more well-known and documented
cases, and covers the evolution of this science and its increasing applications. At the
end, the past becomes prolog as the next steps in the progression of this science are
discussed and the implications it will have on the ability to identify the missing.
The Armed Forces DNA Identification Laboratory (AFDIL) was established in the
Washington DC area in the early 1990s to initially aid in the identification of missing
US service members from Vietnam and I relocated to this laboratory in 1994, where
I would become the Chief of Staff of the mtDNA Section. The Central Identification
Laboratory, Hawaii (CILHI) would locate and recover remains believed to belong
to missing US service members and establish a presumptive identification based
upon document review and crash site investigation as well as an examination of the
remains. This often led to a presumption of the identification of the remains, but
not necessarily the conclusive proof of identity. The main role of mtDNA testing
was to provide supporting evidence for this presumptive identification and mtDNA
testing came at the end of the identification process. At that time, there were several
universities that had also begun to develop and apply this type of testing to skeletal
remains.
Once a person dies, their DNA begins to degrade and since mtDNA is present in
dozens to thousands of copies per cell, mtDNA sequence fragments will be present
after nuclear DNA becomes too degraded to test. Thus, mtDNA was most often
used with skeletal remains testing in the early days of DNA testing skeletal remains.
However, as mtDNA is maternally inherited, it does not point toward individuality,
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Huffine Global Solutions, Lawton, USA

https://doi.org/10.1007/978-981-19-0043-3_3
26 E. Huffine
but rather a maternal line. The process of obtaining mtDNA is both expensive and
time consuming compared to testing for nuclear DNA. In the early days of AFDIL,
from the mid to late 90s, a team of two scientists would work to test 3–5 bone
samples per month, as well as a varying number of family references. AFDIL had a
dedicated QA/QC section that would ensure the reagents were of sufficient quality
and contamination free and track the maintenance of equipment and the expiration
date of reagents. The quality of work performed by AFDIL was recognized as world
leading in mtDNA testing and scientists from other nations would come to AFDIL
and be trained in several DNA testing techniques. In this way, AFDIL ‘seeded’ much
of the world’s forensic DNA community in the testing of skeletal remains.
In 1997, AFDIL obtained mtDNA profiles from the remains in the Tomb of the
Unknown Soldier, Vietnam and this provided supporting evidence in the identifi-
cation of these remains as belonging to Michael J. Blassie [1]. Given the evolving
and increasing power of DNA testing and other forensic science, it was determined
that this tomb would remain empty as it was believed any other remains placed into
the Tomb may one day be identified. The early successes of AFDIL in aiding in
the identification of the missing from Vietnam led to an expansion of the AFDIL
mission to include the mission from the Korean War and World War II. The AFDIL
team expanded staffing and equipment to better accommodate the expanding number
of cases that could be tested and this mission continues. In early 1999, one of my
staff members showed me a flyer outlining the need for DNA testing in the former
Yugoslavia to aid in the identification of tens of thousands of missing. Obviously, the
rate of identifications based upon mtDNA would be far too slow to have a significant
impact on this number of missing and new applications of forensic DNA testing
would need to be developed.
The breakup of the former Yugoslavia resulted in tens of thousands of dead and
missing. As outlined above, to this point in history DNA testing had only been
used at the end of the identification process, after other forensics and investigative
techniques had developed a presumption of identity. Once such a presumption of
identity had been developed, relatives would be located and a DNA sample taken
from them to compare to the DNA profile obtained from remains. This type of
process in a city called Tuzla in Bosnia had led to 7 identifications of 4000+ bodies
over a 3-year period. This was woefully slow and would not serve the purpose
of mass identifications, and for DNA testing to provide a greater role and aid in
the identification of tens of thousands, new procedures and process flow protocols
would need to be developed. I was offered a one-year contract to review and make
recommendations on how, or if, DNA testing could be applied in the context of mass
identifications in Bosnia and I resigned my position as the Chief of the mtDNA
Section at AFDIL and moved to Sarajevo, Bosnia and Herzegovina in April of 1999.
As with many endeavors in life, the path forward was not simple. When I resigned
my position as the Chief of Section at AFDIL to head to Bosnia, virtually none of
my colleagues believed what I would be attempting was possible and I was even told
by some that I was committing professional suicide.
A short time after accepting the position in Bosnia and resigning from AFDIL,
the US and NATO began a bombing campaign of Serbia forces due to activities in
3 Inscription from Author’s Diary 27
Kosovo. The Sarajevo airport was closed and against this backdrop of uncertainty, I
flew to the former Yugoslavia. As the Sarajevo airport was closed, I flew into Zagreb,
and was driven to Sarajevo. As in developing any new project, most of the initial
challenges were centered on understanding the issues and current capabilities and
resources that could be applied to providing recommendations for the project. In the
case of Bosnia, in addition to the creating DNA laboratories and training staff, well
over 10,000 bodies still needed to be recovered, funding needed to be raised, and, as
blood samples would need to be obtained from the families of the missing, a complex
situation existed to be solved. These are factors that were in addition to the science
of DNA testing not yet being capable of providing large-scale DNA identifications.
As one example of this complexity, in the city of Tuzla, more than 4000 body bags
containing remains of the missing from Srebrenica were located. Many of these body
bags contained commingled remains, meaning that in some cases, one body bag may
contain the remains of more than one person. In addition, one person’s remains
may be contained within more than one body bag, adding to an already complex
identification situation as there was a need for a large-scale reassociation effort. As
anthropological analysis would not be adequate by itself to resolve such a large-
scale identification need, additional DNA testing would be needed for reassociation
of skeletal elements. As mentioned earlier, prior to the application of a DNA-led
project, seven sets of remains had been identified over a three-year period by the
use of ‘classic’ system of identification, in which DNA testing was used at the end
of the identification process to support a presumptive identification. This rate of
identification was far too slow to provide answers to the families of the missing in
a timely manner, or to assist in understanding how and where mass executions were
carried out.
The International Commission on Missing Persons (ICMP) was created during the
G-7 summit in Lyon, France in 1997 and was designed to be a blue-ribbon commis-
sion to address several issues, among of which was the recovery and identification
of the missing persons in former Yugoslavia [2]. The application of using DNA
testing to assist in the identification process was viewed as providing at least a partial
solution to the identification process, but to do so in the numbers of identifications
needed, a fundamental shift in its application would be needed. The use of mtDNA
testing would not be adequate for such large-scale DNA testing. For mass DNA-led
identifications to be performed on tens of thousands of sets of remains, not only
would a reliable method for obtaining nuclear DNA from skeletal remains need to be
developed, but the entire identification strategy would need to be altered. Beginning
in the mid to late 90s, both Slovenia and Croatia had started performing DNA testing
using nuclear DNA in their identification efforts, thus showing that a strategy based
upon using nuclear DNA testing instead of mtDNA testing might be feasible. This
led to what was at that time a bold strategy to mass DNA test all the remains and
collect DNA samples from all relatives who were missing loved ones and compare
these profiles to each other.
Upon my arrival into Sarajevo, much of my initial efforts focused on educating
local political leaders, family groups of the missing, and potential donors as to the
potential of DNA testing to aid in the identification of thousands. During this time,
28 E. Huffine
it became apparent that there remained strong emotions among the primary ethnic
groups that resided in Bosnia that presented challenges in creating the integrated
type of system needed to maximize success. The remains of many of the missing
were scattered in across the country and across ethnic divisions and family members
were frequently located in different regions than the remains of their missing family
members. In some cases, bodies had been thrown into rivers that took the bodies
across national borders. Furthermore, many medical and other records had been
destroyed during the war, or were not updated. Mass graves were still being located
and exhumed by international forensic teams and in some cases, the information
obtained from these teams were to be used for charges of wars crimes and crimes
against humanity, which could impact the willingness of some leaders to support
large scale DNA identifications. Once a body is identified and the name returned
to that body, the history of the person is returned, including those with whom they
were last seen and the events that happened to them immediately prior to their death
may be revealed. While this is powerful for a single identification, when multiplied
by hundreds or thousands, patterns become apparent which is invaluable evidence
in determining motive and large-scale operations. As mentioned previously, in some
cases the bodies were badly commingled and this most often occurred with the
missing of the estimated 8000 mostly men and adolescence boys from the city of
Srebrenica. The bodies of these missing were buried in primary mass graves, and
then these primary mass graves would be dug up, often by heavy machinery, and
placed into secondary mass graves. These secondary mass graves were sometimes
made by combining multiple primary mass graves. Sometimes, these secondary mass
graves were dug up and the bodies moved to tertiary mass graves. This process
severely disarticulated the remains and commingled the bodies and this occurred
with thousands of the missing.
The concept of a DNA-led identification process, if successfully created, would
fundamentally alter the role of DNA testing in the identification process. MtDNA
testing would not be adequate to take the lead in making thousands of identifications
due to time and cost considerations as well as its inability to point to individuality. At
that point, most identifications of skeletal remains involved mtDNA being used as a
confirmation tool for a presumptive identification. For mass DNA-led identifications
to be done on thousands of sets of remains, not only would a reliable method for
obtaining nuclear DNA from skeletal remains need to be developed, but the entire
identification strategy would need to be altered. This presented both a scientific as
well as an ‘institutional’ challenge as many in the identification field were resistant
to the dramatic change this represented.
After arriving in Bosnia and beginning to review the on-the-ground realities
that included the number and condition of the missing, limited equipment and
training, insufficient funding, and disagreements among the family organizations
of the missing as well as international organizations, it quickly became apparent that
the scientific modifications to use nuclear DNA as the lead identification method was
only part of challenges. Each of these challenges needed to be solved if a new system
that could identify thousands of missing would be possible. As the local population
as well as international supporters and donors would need to understand how DNA
testing could lead to produce thousands of identifications, a considerable amount of

time during my first year in Bosnia was dedicated to an educational campaign where I
would address family groups, meet with various political figures, and be interviewed
on local news outlets. At that time former Senator Bob Dole was the head of the
ICMP and he visited Bosnia several times, along with other members of the ICMP
oversight committee. At that time, the majority of the funding received by the ICMP
came from the United States and the Netherlands, but it was not dedicated for DNA
use and the ICMP was considered to be non-operational in terms of having an active
role in the recovery and identification process. Additional funding was secured and
equipment donations of sequencers and other materials were made, helping to pave
the way for training and research needed to improve on the ability to obtain nuclear
DNA profiles from skeletal remains and to begin putting the pieces together for a
DNA-led identification system and the ICMP became operational.
We hired young, local staff as well as brought in outside DNA testing experts
and began developing the nuclear DNA testing process. As previously stated, both
Slovenia and Croatia had begun DNA testing of their missing from the breakup of
the former Yugoslavia and were applying various techniques. Laboratories in both
nations, as well as AFDIL, offered training on DNA testing techniques to Bosnian
staff, which we thankfully accepted. In order to overcome some of the residence for
samples being sent across ethnic lines for testing, a testing strategy was developed in
which multiple DNA testing laboratories were established that could only perform
part of the overall testing needed to produce an identification. For example, the labo-
ratory in Sarajevo would only be able to process skeletal remains. The laboratory
in Tuzla would only process blood samples for STR testing. The other laboratories
in Banja Luka and Belgrade would only be able to perform specialized testing for
Y-STR and mtDNA testing as well as coordinate research and development of new
procedures and the writing, review, acceptance, and implementation of SOPs. Proto-
cols for which skeletal elements most likely contained the highest levels of DNA
and how to sample those elements for DNA testing were written and distributed
to the mortuaries across Bosnia. In addition, a strategy for the collection of blood
samples via a blood stain from a finger prick was developed and implemented. This
strategy consisted of both fixed collection centers set-up in strategic locations as
well as mobile systems. Later, an outreach to other nations that had diaspora from
the former Yugoslavia was untaken to collect samples from family members who
were no longer living in the former Yugoslavia. All cuttings from bone samples as
well as the blood samples collected from families were sent to a central facility in
Tuzla where they were entered into a database, and ‘blinded’ by being transferred
into a container that only had a barcode for identification. This blinding of samples
meant the laboratories did not know from whom or from where the sample origi-
nated. In this way, the foundation of what would become the world’s largest and most
successful DNA testing system for the identification of skeletal remains was being
laid.
It was critically important for the success of this developing DNA-led identifica-
tion system to be able to obtain nuclear DNA from skeletal remains. To date, this
type of success rate in other laboratories was often far less than 50% and a much
30 E. Huffine
higher success rate was needed. By developing and validating new extraction proce-
dure for getting DNA out of skeletal remains, and by using STR kits from Promega,
a robust system for obtaining STR profiles from skeletal remains at more than a
95% success rate was developed. The DNA profiles obtained from skeletal elements
were stored in one database and those from blood references in another. A DNA
matching program was written that would compare the DNA profiles between the
two databases, searching for potential familial matches. If a potential match were
found, other relatives to the developing presumptive identification were sought out
for DNA testing, if needed. Much of the initial testing focused on cases in which
some circumstantial evidence existed, such as information that could indicate to
which group of missing a set of remains might belong. It was hoped this initial
focused effort would result in early identifications while more reference samples
were being collected, tested, and entered into the family reference DNA database.
The power of a DNA database is directly proportional to the size of the database
and the percentage of the missing who are represented by having familial references
in the database. In this way, DNA testing of skeletal elements and blood references
from family members began in late 2001 and DNA profiles began to populate the
databases.
Once data was obtained from skeletal and family references, it was sent to the
central facility in Tuzla, Bosnia. The data that was received from across the nation
was then entered into the local database and a copy of the profiles were sent to
the ICMP headquarters located in Sarajevo via a driver delivering the profiles on a
computer floppy disk as the databases were not placed onto the Internet. It was at
the Sarajevo headquarters that profiles were compared. On November 16th, 2001,
which happened to be the first day of Ramadan that year, the day’s profiles had been
hand delivered and several of my staff and I gathered around the computer screen
as profiles from skeletal remains and family references were being compared. The
sun had already set on that day and the glow from the computer screens produced a
faint blue light in the room. We found a match between a bone sample and a husband
and wife who were missing a son. At that moment, I knew history had been made
and an entire science had been forever altered. This match would be to a 17-year-old
boy, killed during the fall of Srebrenica. Another match was made later that night, to
a 15-year-old boy. The next day, we informed the mortuary that had submitted the
bone sample for testing of the matches. The mortuary pulled these two body bags
and examined the remains. These remains were consistent with the pre-mortem data
of these two individuals as well as the circumstantial evidence associated with these
remains. At that moment, the mortician also realized that a science had been changed
and that DNA matches provided them the information they needed as it gave them
a presumptive identification in which they then used other forensic techniques to
confirm the identification. The role of DNA testing in the identification process of
skeletal remains was switched as it now took the lead in the identification process.
The moment of this first in-country DNA identification would become a national
day of remembrance of the missing in Bosnia and would later be noted in Times
‘Timelines of History’ as a major scientific achievement.
There is an old adage that success brings success, and this was true with this
system proving itself. As news about these first identifications was released, more
family members who were missing loved ones would donate reference samples,
increasing the power of the DNA database. For that first month, 5 identifications
were made. The following month, 7 identifications were made. Within 6-months,
200+ identifications were being made per month and after one year, more than 500
DNA matching reports were being generated per month. As the DNA database of
family references grew, the matching rate also grew to the point that an exhumed set
of remains would have more than an 80% chance of being identified by this system.
As the operation of the DNA-led identification system continued, lessons were
learned and applied in the unique context of Bosnia, but that could be applied to
other forensic DNA identification systems. One example of this is using the newly
identified bodies as references to identify other missing family members. In the
Bosnia, it was not uncommon for a family to be missing multiple family members.
Using skeletal remains to help identify the other missing family members became a
relatively common occurrence in Bosnia. For severely commingled remains, espe-
cially those associated with Srebrenica mass graves that were dug up and moved
during the war, an initial strategy to test each femur, humorous, skull, and the most
commonly represented skeletal element in a body bag that contained commingled
remains was developed. This provided insights into the minimum number of individ-
uals represented in the body bag, as well as their identification. This also provided
the mortuary with an understanding of which bodies may be associated with other
bodies in other body bags, thus providing additional information that could be used
to associate certain body bags with each other based upon the identity of missing
they contained. Once the full STR profiles were obtained on these skeletal elements
by using the Promega kits, a subset of prime sets that corresponded to some of these
STR loci were used to link other skeletal elements to those bone samples that had
provided a full STR profile. The mortuary staff had the ability to submit samples from
any additional bone sample for testing as they attempted to reassociate disarticulate
remains. It was understood that even this refinement of testing would not be able to
provide DNA profiles for all skeletal elements as the time and cost to do so would be
prohibitive. Discussions were held with the families of the missing and agreements
reached on this testing strategy. In the case of Srebrenica, for those elements that did
not undergo DNA testing and in which the anthropologist/mortuary staff could not
link to a body, it was agreed those remains would be placed into a special building
to remember the missing. Many of these lessons learned were presented as papers or
presentations at the 2002 American Academy of Forensic Sciences annual meeting
in February. There were a total of 13 such presentations given by ICMP staff at
this conference, which represented the most given by any one organization. In this
way, these lessons and solutions were shared with others around the world and this
new DNA-led identification system begun to spread and would become the template
example for future mass DNA-led identification systems [3].
On September 11th, 2001, the attack on the World Trade Center, the Pentagon, and
the plane crash in Pennsylvania meant there were thousands of mostly Americans
who needed to be identified. Though the system at the ICMP was about two months
32 E. Huffine
from producing its first identification, its considerable research and development
had been on-going in Bosnia. Three staff members from the ICMP were flown to
New York City to advise New York and US officials on what had been learned
in Bosnia in the development of a system to identify thousands of missing. The
DNA testing for these samples was performed in several laboratories, including the
Bode Technology Group and would involve the full range of DNA testing of STRs,
Y-STRs, and mtDNA. New laboratory information management systems (LIMS)
DNA specific computer programs were developed as a result of this need to identify
thousands of missing by using DNA testing.
Based upon the lessons learned in the former Yugoslavia from the DNA-led identi-
fication system, as well as those learned from 9 to 11, several NGOs in Latin America
agreed to work together to launch the Latin American Initiative for the Identification
of the Disappeared. Representatives from Peru, Guatemala, and Argentina meet in
Washington DC to lobby Congress and other US based NGOs to further the aware-
ness of what happened in Latin America as well as seek funding for DNA testing
within their nations. Each of these nations experienced loss of life due to internal
conflicts and how people were ‘disappeared’ varied among the nations. This lobbying
effort was successful and funding was provided to these NGOs to engage in the loca-
tion, recovery, examination, document review, and DNA testing of cases from their
nations. At first, both family reference and skeletal samples were outsourced to
The Bode Technology Group for testing. DNA profiles were obtained from the vast
majority of skeletal samples and as the DNA database of family references grew,
the number of identifications also grew. To demonstrate the power of DNA testing,
the identifications of thousands of missing Argentineans from the 1970s ‘Dirty War’
lead to charges against those responsible, even though these events had happened
decades earlier. This shows the power of DNA testing: it can reveal the identities of
the missing or the biological ownership of samples decades later [4].
Some of the funding for the Latin American Initiative for the Identification of
the Disappeared went improving an existing DNA laboratory in Argentina and some
went to aid in the creation of a DNA laboratory in Guatemala. In this way, local
expertise in these nations was developed. Additional missing persons projects were
established for El Salvador, Mexico, and other nations, all of which were based
upon the Bosnia model. A DNA-led system is now being used to help provide the
identifications for many mass losses of life incidents from natural and manmade
disasters around the world as well as for individual identifications from recovered
bodies. As the understanding of DNA testing and the power DNA databases grew,
additional applications of this science developed.
References
1. https://www.military.com/history/air-force-1st-lt-michael-blassie.html.
2. https://www.icmp.int/where-we-work/europe/western-balkans/bosnia-and-herzegovina/.
3. https://pubmed.ncbi.nlm.nih.gov/11387637/.
4. https://www.orlandosentinel.com/news/os-xpm-2006-06-11-latam11-story.html.
Chapter 4
DNA Sample Collection and Packaging:
Chain of Custody
DNA Sample Identification and Its Integrity in Crime Scene
Any biological or non-biological elements that are expected to be useful in analyzing

the case are considered as evidence in the crime scene. Most preliminary step in
forensic investigation of any case is to reach the crime scene and analyze the situation,
collect the samples unaltered and without contaminating. The aim is to collect all
the samples and compare their DNA profile with the known profiles of suspects. At
most care is taken to prevent tampering and mishandling of evidence. Though all the
biological material is collected for DNA profiling sometimes even the non-biological
material like used cigarette butt, used coffee cup, toothpick, etc. can also be useful
for DNA study using touch DNA concept. Thus it becomes indispensable to collect
all possible evidence from the scene retaining their quality and integrity.
The commonly identified biological samples in the crime scene include blood,
hair, tissue, semen on the clothes or from victim, etc. There are specialized kits for
collecting such samples to retain the quality of evidence during transit from crime
scene to forensic lab.
Contamination and Elimination Sample
In the early days of DNA testing, there needed to be a sufficient sample of genetic
material to be tested, which by today’s standard, required a significantly larger
sample. The discovery and application of the polymerase chain reaction (PCR)
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36 A. Kumar and A. Kashyap
markedly decreased the amount of DNA required for testing and greatly expanded the
number of samples in which DNA testing could be applied. The PCR technique adds
a mix of enzymes, proteins, bases, and other materials to DNA that has been isolated
from a sample. This mix then undergoes a series of heating and cooling cycles. During
each cycle, a selected region of DNA is amplified and the targeted DNA undergoes a
geometric increase in its number of units by doubling each cycle. Thus, one sequence
of DNA becomes two identical sequences after one heating/cooling cycle, which then
becomes four identical sequences after another cycle, which then becomes 8, 16, 32,
etc. One sequence of DNA can end up creating millions of identical copies of itself
within 60–90 min and this dramatic increase in its number permits it to be detected
by a variety of instruments [1].
This ability to force replication of DNA sequences millions of times introduces
concerns about contamination from other sources of DNA as those sources may
provide additional target DNA molecules that would be co-amplified. Such co-
amplification can lead to an incorrect DNA profile being assigned to a sample and
precautions need to be taken in the collection, storage, and laboratory settings to
reduce the chance of contamination. Special training of those who collect crime
scene samples for DNA testing should be part of any collection efforts. Labora-
tories need to undergo strict validation procedures that comply with ISO 17025
standards for DNA testing and regular use of controls for all testing must be standard
[2]. Furthermore, all those who are involved in the collection and testing of DNA
samples need to have their DNA profiles as part of a contamination DNA database
that profiles obtained from samples can be compared against to eliminate contamina-
tion from staff as a possibility. Ideally, all profiles generated in the laboratory would
be contained in an internal laboratory DNA database to help detect sample-to-sample
contamination. However, this type of contamination database may be forbidden by
state and/or federal laws against a laboratory maintaining DNA profiles from crime
scenes and/or database samples. It should be noted that the most likely result of
reporting a contaminate as the profile obtained from a sample would result in a false
exclusion.
The samples collected from crime scenes are random and present in harsh envi-
ronmental conditions prior to their collection. There are high chances of its contam-
ination with external DNA/ microbial cells/ dirt particles making the sample lose
its purity. All of these hinder the quality and quantity of DNA extracted. Thus a
greater degree of attention is necessary while collecting, packaging, and transit of
the evidence. A simple sneeze, cough, or contaminated hand can be a high-end risk
for sample integrity.
Eliminations samples are all those which are collected purposefully from the
individual who had legitimate access to crime scene objects. Common elimination
samples include DNA fingerprint, DNA buccal swab, etc. Elimination samples play a
key role in categorizing the crime scene samples as from the criminal/suspect or any
other individual present in the scene. For instance, in a burglary case, the possibility
of finding the suspect sample is as high as the samples of the housemates. Thus
it is necessary to eliminate the housemate profiles as elimination samples in DNA
comparisons [3].
4 DNA Sample Collection and Packaging: Chain of Custody 37
Sources to Obtain DNA from Crime Scene
Chances of obtaining DNA evidence from crime scene may be anything without
restriction. Even a collected dental impression mold or a tooth bite on victim, half-
eaten fruit, etc. can act as a source of DNA in identification of individuals. DNA is
expected to be obtained from blood, semen, organs, bones, teeth, skin cells, muscle,
brain cells, hair, saliva, mucus, perspiration, fingernails, urine, feces, etc. It is the
combined effort of investigating agency and laboratory person in identifying and
collecting potential samples from crime scene.
It is possible to collect DNA from blood, skin cells, semen, hair roots, urine,
and saliva. The types of materials used for DNA analysis are blood and bloodstains,
semen and semen stains, bones, teeth, hair with root, hair shaft, saliva, urine, pieces,
debris from fingernails or broken fingernails, muscle tissue, cigarette buds, postage
stamp (licked), envelope ceiling flaps, dandruff, fingerprints, personal items like
razor blade, chewing gum, wristwatch, ear wax, toothbrush, clothes, used tools, used
toothpick, bite marks, etc.
Physical evidence collected from crime scene can either link a suspect to the crime
scene or eliminate him just like fingerprints. The direct transfer of DNA from one
individual to another individual or to an object can be used to link a suspect to a
crime scene. This direct transfer could involve:
The suspect’s DNA posted on the victim’s body or clothing
The suspect’s DNA deposited on an object
The suspect’s DNA posted at the location
The suspect’s DNA deposited on the suspect’s body or clothing
The suspect’s DNA deposited on an object
The suspect’s DNA posted at a location
The witness’s DNA poster on victim’s or suspect
The witness’s DNA deposited on an object or at a location.
DNA evidence collection from a crime scene must be performed carefully and a
chain of custody established in order to produce DNA profiles that are meaningful
and legally accepted in court. DNA testing techniques have become so sensitive that
biological evidence too small to be easily seen with the naked eye can be used to
link suspects to crime scenes. The evidence must be carefully collected, preserved,
stored, and transported prior to any analysis conducted in a forensic DNA laboratory.
Sample Collection for DNA Profiling
Sample collection, though a simple technical term, has broad horizons. There are
different types of samples that can have different collection protocols. Commonly
used samples for DNA profiling include samples collected from individuals person-
ally include Fresh blood and saliva. Samples collected from the victim or crime scene
38 A. Kumar and A. Kashyap
Fig. 4.1 DNA evidence

collection kit. Picture
Courtesy Sirchie catalog
include Blood Stain, Semen sample, Bone remnants, Body tissues, used tooth bud,
cigarette butt, etc. all of which have a different collection protocol.
There are also some specialized kits for the collection of crime scene samples that
not only allows easy collection but also enable contamination-free transit to the lab.
Some of the commercially available DNA sample collection kits include Bode Tech-
nology Collection kit, DNA Pro Swab collection kit, Forensic DNA Fingerprinting
Kit, Blood and Urine collection kit, etc. The kit may generally contain Gloves,
Forceps, Sterile containers, Swabs, Buffer, Syringes and needles, Blood Collection
tubes, etc. [4] (Fig. 4.1).
Blood—3–5 ml of blood should be collected in sterile vial with EDTA.
Blood-stained clothes—Blood-stained garments should be thoroughly air-dried
and packed in separate paper packets.
Blood-stained articles or weapons—In case the stains are dry, scrape the stained
area, keep in paper envelope and seal or swab the stains with a sterile gauze cloth
soaked with saline; dry and pack separately. Stains on immovable services such as
tables or floors may be transferred with sister oil cotton swabs and distilled water.
Rub the stained area with the moist swab until the stain is transferred to the swab.
Allow the swab to air dry without touching any others. Store each swab in a separate
paper envelope.
Semen or seminal stains or vaginal swabs—Air dry and pack in separate paper
packets.
Hairs—Hair with roots to be air-dried and packed in separate paper packets.
Tissue of skin, muscle, etc.—Muscle tissues to be collected in clean glass bottle
with 20% Dimethyl sulphoxide or Sodium chloride.
Bones—Whole bones like femur and humerus are to be collected and packed in
separate paper packets and kept in aluminum foil and frozen.
Teeth—Teeth should be packed in dry paper packets and sealed (Molar/canine
are preferred).
Visceral matter—Collect intestine, spleen, and heart (Avoid liver).
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The imperial princes of the blood, all nearly related to the Emperor,
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Five of these ten princely houses are the old Gosekke, the first five
of the one hundred and fifty-five kugé families comprising the old
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Takatsukasa, Nijo, and Konoye families, rank, since 1883, the
houses of Sanjo, Iwakura, Shimadzu, Mori, and Tokugawa, sharing
with them the privilege of offering the bride to the heir-apparent.
The Emperor visits personally at the houses of these ten princes,
and recently the Tokugawas entertained him with a fencing-match
and a No dance in old style, the costumes and masks for which had
been used at Tokugawa fêtes for centuries. In accordance with other
old customs, a sword by a famous maker was presented to the guest
of honor, and a commemorative poem offered in a gold lacquer box.
Yet the head of the Tokugawa house is a grandson of the Shogun
who first refused to treat with Commodore Perry, and son of Keiki,
the arch rebel and last of the Shoguns, who for so long lived
forgotten as a private citizen on a small estate near Shidzuoka,
keeping alive no faction, awaking no interest—attaining, in fact, a
political Nirvana.
Under new titles the old fiefs are lost sight of and old associations
broken up. The marquises, counts, and barons of to-day are slender,
dapper little men, wearing the smartest and most irreproachable
London clothes, able to converse in one or two foreign languages on
the subjects that interest cosmopolitans of their rank in other
empires, and are with difficulty identified with their feudal titles. The
Daimio of Kaga has become the Marquis Maeda, his sister married
the Emperor’s cousin, and the great yashiki of their ancestors has
given way to the buildings of the Imperial University. The Daimio of
Satsuma is now Prince Shimadzu. It is not easy to associate these
modern men-about-town, who dance at state balls, who play billiards
and read the files of foreign newspapers at the Rokumeikwan, who
pay afternoon calls, attend teas, garden-parties, dinners, concerts,
and races; who have taken up poker and tennis with equal ardor,
and are victimized at charity fairs and bazaars, with their pompous,
stately, two-sworded, brocade and buckram bound ancestors.
There are great beauties, favorites, and social leaders among the
ladies of the court circle, and the change in their social position and
personal importance is incredible. Japanese matrons, who, a few
years ago, led the most quiet and secluded existence, now preside
with ease and grace over large establishments, built and maintained
like the official residences of London or Berlin. Their struggles with
the difficulties of a new language, dress, and etiquette were heroic.
Mothers and daughters studied together with the same English
governess, and princesses and diplomats’ wives, returning from
abroad, gave new ideas to their friends at home. Two Japanese
ladies, now foremost at court, are graduates of Vassar College, and
many high officials are happily married to foreign wives; American,
English, and German women having assumed Japanese names with
their wedding vows. The court has its reigning beauty in the wife of
the grand master of ceremonies, the richest peer of his day, and
representative of that family which gave its name to the finest
porcelain known to the ceramic art of the empire.
Tokio society delights in dancing, and every one at court dances
well. Leaders of fashion go through the quadrille d’honneur, with
which state balls open, and follow the changes of the lancers with
the exactness of soldiers on drill, every step and movement as
precise and finished as the bending of the fingers in cha no yu. The
careless foreigner who attempts to dance an unfamiliar figure
repents him of his folly. Japanese politeness is incomparable, but the
sedateness, the precision, and exactness of the other dancers in the
set will reproach the blunderer until he feels himself a criminal. The
ball is the more usual form of state entertainments. The prime-
minister gives a ball on the night of the Emperor’s birthday, and the
governor of Tokio gives a ball each winter. From time to time the
imperial princes and the ministers of state offer similar
entertainments, and every legation has its ball-room. The members
of the diplomatic corps are as much in social unison with the higher
Japanese circles as it is possible to be with such subjects at any
capital, and the round of tiffins, dinners, garden-parties, and small
dances makes Tokio very gay during the greater part of the year.
The first formal visiting of the season begins in October, and by
May social life is at an end until hot weather is over. Lent makes little
break in the social chain. Great seriousness and exactness in social
usage is inherent in this high-bred people. Visits of ceremony are
scrupulously paid within the allotted time, and a newly-arrived official
in Tokio finds no diminution of the card-leaving and visiting which
awaits him in any other capital. At the houses of the imperial princes
cards are not left, the visitor inscribing his name in a book in the hall.
After each state ball, a guest must call at once upon the princess, or
minister’s wife, who presided, and any remissness strikes his name
from her list.
Garden-parties are the favorite expression of Tokio hospitality. All
official residences in the city have fine grounds, and many ministers
of state own suburban villas. A few of the legations are able to
entertain in the same way, and many military officers make the
garden of the old Mito yashiki, now the Arsenal grounds, the scene
of their courtesies.
There is a stately court journal, which is the official bulletin, but
Tokio has not yet set up a paper of society gossip and scandal for
the rigorous censorship of the Japanese press to expunge; nor are
there books of court memoirs. Yet what memoirs could be more
interesting than those that might be written by the men and women
who were born in feudal times, who have lived through the exciting
days of the Restoration, and have watched the birth and growth of
New Japan.
CHAPTER XIII
THE SUBURBS OF TOKIO
The suburbs of Tokio are full of holiday resorts for the people and
the beautiful villas of nobles. To the north-east, in Oji, are the
Government chemical works and paper mills, where rough bits of
mulberry-wood are turned into papers of a dozen kinds, the silkiest
tissue-paper, smooth, creamy writing-paper, thick parchment, bristol-
board, and the thin paper for artists and etchers. On a sheet of the
heaviest parchment paper I once stood and was lifted from the floor,
the fabric showing no mark of rent or strain, and it is wellnigh
impossible to tear even a transparent Oji letter sheet. The Oji tea-
house has a famous garden, and in autumn Oji’s hill-sides blaze with
colored maples, and then the holiday makers mark the place for their
own.
Waseda, the northern suburb, contains an old temple, a vast,
gloomy bamboo-grove, and the villa of Countess Okuma, to whose
genius for landscape-gardening is also due the French Legation’s
paradise of a garden, in the heart of the city, that place having been
Count Okuma’s town residence before he sold it to the French
Government. From Waseda’s rice fields a greater marvel grew.
Meguro, south of Tokio, is a place of sentimental pilgrimage to the
lovers of Gompachi and Komurasaki, the Abelard and Heloise of the
East, around whose tomb the trees flutter with paper poems, and
prayers. In the temple grounds are falling streams of water, beneath
which, summer and winter, praying pilgrims stand, to be thus
pumped on for their sins. Similar penitents may be seen at a little
temple niched in the bluff of Mississippi Bay. Meguro has an annual
azalea fête and a celebration of the maple-leaf, and its resident
nobles, among whom is General Saigo, give feasts in honor of the
season’s blooms.
The Sengakuji temple, near Shinagawa, is a sacred spot and
shrine of chivalry, the burial-place of the Forty-seven Ronins; and
here come pious pilgrims to say a prayer and leave a stick of burning
incense, and view the images and relics in the little temple.
Near Omori, half-way between Yokohama and Tokio, Professor
Morse discovered the shell-heaps of prehistoric man. The
neighborhood is made beautiful by old groves, old temples and
shrines, tiny villages, picturesque farm-houses, and hedge-lined
roads, while Ikegami’s temples shine upon the hill that stands an
evergreen island in the lake of greener rice fields or golden stubble.
Here died Nichiren, founder of the Buddhist sect bearing his name.
For six centuries these splendid temples have resounded with the
chantings of his priesthood, who still expound his teachings to the
letter. The Nichiren sect is the largest, richest, most influential, and
aggressive in Japan. They are the Protestants and Presbyterians of
the Buddhist religion; firm, hard, and unrelenting in their faith,
rejecting all other creeds as false, and zealously proselyting.
Nichiren was a great scholar, who, poring over Chinese and Sanscrit
sutras, believed himself to have discovered the true and hidden
meaning of the sacred books. His labors were colossal, and though
exiled, imprisoned, tortured, and condemned to death, he lived to
see his followers increasing to a great body of true believers, and
himself established as high-priest over the temples of Ikegami. In the
popular play “Nichiren,” one has thrilling evidence of what the pious
founder and his disciples endured.
On the twelfth and thirteenth of each October special services are
held in memory of Nichiren, which thousands of people attend. On
the first day of this matsuri the railway is crowded with passengers.
Bonfires and strings of lanterns mark the Omori station by night, and
by day the neighboring matsuri is announced by tall bamboo poles,
from which spring whorls of reeds covered with huge paper flowers.
These giant flower-stalks are the conventional sign for festivities, and
when a row of them is planted by the road-side, or paraded up and
down with an accompaniment of gongs, the holiday spirit responds
at once. The quiet country road is blockaded with hundreds of
jinrikishas going to and returning from Ikegami’s terraced gate-ways.
Men, women, and children, priests, beggars, and peddlers pack the
highway. The crowd is amazing—as though these thousands of
people had been suddenly conjured from the ground, or grown from
some magician’s powder—for nothing could be quieter than Omori
lanes on all the other days of the year.
Along the foot-paths of the fields women in tightly-wrapped
kimonos with big umbrellas over their beautifully-dressed heads;
young girls with the scarlet petticoats and gay hair-pins indicative of
maidenhood; little girls and boys with smaller brothers and sisters
strapped on their backs, trudge along in single files, high above the
stubble patches, to the great matsuri. In farm-house yards
persimmon-trees hang full of mellow, golden fruit, and the road is
literally lined with these apples of the Hesperides. Peddlers sit on
their heels behind their heaped persimmons and busily tie straw to
the short stems of the fruit, that the buyer may carry his purchase
like a bunch of giant golden grapes. Fries, stews, bakes, and grills
scent the air with savors, and all sorts of little balls and cubes, pats
and cakes, lumps and rolls of eatables are set out along the country
road. A queer sort of sea-weed scales, stained bright red, is the
chewing-gum of the East, and finds a ready market.
On the days of the matsuri the village street is impassable, and the
whole broad walk of the temple grounds leading from the pagoda is
lined with booths, jugglers, acrobats, side-shows, and catch-penny
tricksters. The “sand-man,” with bags of different colored sands,
makes beautiful pictures on a cleared space of ground, rattling and
gabbling without cessation while he works. First he dredges the
surface with a sieve full of clean white sand, and then sifts a little thin
stream of black or red sand through his closed hand, painting
warriors, maidens, dragons, flowers, and landscapes in the swiftest,
easiest way. It is a fine example of the trained hand and eye, and of
excellent free-hand drawing. A juggler tosses rings, balls, and knives
in the air, and spins plates on top of a twenty-foot pole. His colleague
balances a big bamboo on one shoulder, and a small boy climbs it
and goes through wonderful feats on the cross-piece at the top. A
ring of gaping admirers surrounds a master of the black art, who
swallows a lighted pipe, drinks, whistles, produces the pipe for a puff
or two, swallows it again, and complacently emits fanciful rings and
wreaths of smoke. Hair-pins, rosaries, toys, and sweets are
everywhere for sale.
A huge, towering, heavy-roofed red gate-way admits streams of
people to the great court-yard, surrounded on three sides by temples
large and small, where the priests chant and pound and the faithful
pray, rubbing their rosaries and tossing in their coins. At one shrine
greasy locks of hair tied to the lattices are votive offerings from those
who have appealed to the deity within. There is a little temple to the
North Star, where seamen and fisher-folk pray, and one to Daikoku,
the god of riches and abundance, the latter a fat little man sitting on
bags of rice, and always beset by applicants.
In the great temple pyramids of candles burn, incense rises, bells
sound, and money rains into the big cash-box at the head of the
steps. The splendid interior is a mass of lacquer, gilding, and color,
the panelled ceiling has an immense filigree brass baldaquin
hanging like a frosted canopy over the heads of the priests, and a
superb altar, all images, lotus-leaves, lights, and gilded doors,
dazzles the eye. Under the baldaquin sits the high-priest of the
temple, who is a bishop of the largest diocese in Japan, while at
either side of him more than two hundred celebrants face each other
in rows. The priestly heads are shaven, the smooth faces wear the
ecstatic, exalted expression of devotees purified by vigil and fasting,
and over their white or yellow gauze kimonos are tied kesas, or
cloaks of rich brocade. The lesser hierarchy appear in subdued
colors—gray, purple, russet—but the head priest is arrayed in
gorgeous scarlet and gold, and sits before a reading-desk, whose
books are covered with squares of similar brocade. He leads the
chanted service from a parchment roll spread before him, at certain
places touching a silver-toned gong, when all the priests bow low
and chant a response, sitting for hours immovable upon the mats,
intoning and reading from the sacred books in concert. At intervals
each raps the low lacquer table before him and bends low, while the
big temple drum sounds, the high-priest touches his gong, and
slowly, behind the lights and incense clouds of the altar, the gilded
doors of the shrine swing open to disclose the precious image of
sainted Nichiren. On all sides stand the faithful, extending their
rosary-wrapped hands and muttering the Nichirene’s special form of
prayer: “Namu mio ho ren ge kio” (Glory to the salvation-bringing
book, the blossom of doctrine).
After seven hours of worship a last litany is uttered, and the
procession of priests files through the grounds to the monastery,
stopping to select from the two hundred and odd pairs of wooden
clogs, waiting at the edge of the temple mats, each his proper pair.
The high-priest walks near the middle of the line underneath an
immense red umbrella. He carries an elaborate red lacquer staff, not
unlike a crozier, and even his clogs are of red lacquered wood. The
service in the temple suggests the forms of the Roman Church, and
this Buddhist cardinal, in his red robes and umbrella, is much like his
fellow-dignitary of the West.
To citizens of the United States Ikegami has a peculiar interest.
When the American man-of-war Oneida was run down and sunk with
her officers and crew by the P. and O. steamer Bombay, near the
mouth of Yeddo Bay, January 23, 1870, our Government made no
effort to raise the wreck or search it, and finally sold it to a Japanese
wrecking company for fifteen hundred dollars. The wreckers found
many bones of the lost men among the ship’s timbers, and when the
work was entirely completed, with their voluntary contributions they
erected a tablet in the Ikegami grounds to the memory of the dead,
and celebrated there the impressive Buddhist segaki (feast of hungry
souls), in May, 1889. The great temple was in ceremonial array;
seventy-five priests in their richest robes assisted at the mass, and
among the congregation were the American admiral and his officers,
one hundred men from the fleet, and one survivor of the solitary
boat’s crew that escaped from the Oneida.
The Scriptures were read, a service was chanted, the Sutra
repeated, incense burned, the symbolic lotus-leaves cast before the
altar, and after an address in English by Mr. Amenomori explaining
the segaki, the procession of priests walked to the tablet in the
grounds to chant prayers and burn incense again.
No other country, no other religion, offers a parallel to this
experience; and Americans may well take to heart the example of
piety, charity, magnanimity, and liberality that this company of hard-
working Japanese fishermen and wreckers have set them.
CHAPTER XIV
A TRIP TO NIKKO
The Nikko mountains, one hundred miles north of Tokio, are the
favorite summer resort of foreign residents and Tokio officials. The
railway now reaches Nikko, and one no longer travels for the last
twenty-five miles in jinrikisha over the most beautiful highway,
leading through an unbroken avenue of over-arching trees to the
village of Hachi-ishi, or Nikko.
On the very hottest day of the hottest week of August we packed
our koris, the telescope baskets which constitute the Japanese trunk,
and fled to the hills. Smoke and dust poured in at the car windows,
the roof crackled in the sun, the green groves and luxuriant fields
that we whirled through quivered with heat, and a chorus of
grasshoppers and scissors-grinders deafened us at every halt. At
Utsonomiya it was a felicity to sit in the upper room of a tea-house
and dip our faces and hold our hands in basins of cool spring-water,
held for us by the sympathetic nesans. They looked perfectly cool,
fresh, and unruffled in their clean blue-and-white cotton kimonos, for
the Japanese, like the creoles, appear never to feel the heat of
summer, and, indeed, to be wholly indifferent to any weather. The
same placid Utsonomiya babies, whose little shaved heads bobbed
around helplessly in the blaze of that midsummer sun, I have seen
equally serene with their bare skulls reddening, uncovered, on the
frostiest winter mornings.
Once out of the streets of this little provincial capital, the way to
Nikko leads up a straight broad avenue, lined on both sides for
twenty-seven miles with tall and ancient cryptomerias, whose
branches meet in a Gothic arch overhead. The blue outlines of the
Nikko mountains showed in the distance as we entered the grand
avenue. The road is a fine piece of engineering, with its ascent so
slow and even as to seem level; but at times the highway, with its
superb walls of cryptomeria, is above the level of the fields, then
even with them, and then below them, as it follows its appointed
lines. Before the railway reached Utsonomiya, travellers from Tokio
had a boat journey, and then a jinrikisha ride of seventy miles
through the shaded avenue. This road was made two centuries ago,
when the Shoguns chose Nikko as their burial place, and these
venerable trees have shaded the magnificent funeral trains of the old
warriors, and the splendid processions of their successors, who
made pilgrimages to the tombs of Iyeyasu and Iyemitsu. In our day,
alas, instead of mighty daimios and men-at-arms in coats of mail, or
brocaded grandees in gilded palanquins, telegraph-poles, slim, ugly,
and utilitarian, impertinently thrust themselves forward before the
grand old tree-trunks, and the jinrikisha and the rattle-trap basha
take their plebeian way.
The cryptomeria has the reddish bark and long, smooth bole of the
California sequoia, and through the mat of leaves and branches,
high overhead, the light filters down in a soft twilight that casts a
spell over the place. After sunset the silence and stillness of the
shaded avenue were solemn, and its coolness and the fragrance of
moist earth most grateful. Two men ran tandem with each jinrikisha,
and they went racing up the avenue for ten miles, halting only once
for a sip of cold water before they stopped at the hamlet of Osawa.
The villages, a row of low houses on either side of the way, make the
only break in the long avenue. With its dividing screens drawn back,
the Osawa tea-house was one long room, with only side walls and a
roof, the front open to the street, and the back facing a garden where
a stream dashed through a liliputian landscape, fell in a liliputian fall,
and ran under liliputian bridges. At the street end was a square
fireplace, sunk in the floor, with a big teakettle swinging by an iron
chain from a beam of the roof, teapots sitting in the warm ashes, and
bits of fowl and fish skewered on chopsticks and set up in the ashes
to broil before the coals. The coolies, sitting around this kitchen,
fortified their muscle and brawn with thimble cups of green tea,
bowls of rice, and a few shreds of pickled fish. We, as their masters
and superiors, were placed as far as possible from them, and
picnicked at a table in the pretty garden. After the severe exertion of
sitting still and letting the coolies draw us, we restored our wasting
tissues by rich soup, meats, and all the stimulating food that might
be thought more necessary to the laboring jinrikisha men.
When we started again, with all the tea-house staff singing sweet
sayonaras, a glow in the east foretold the rising moon, and a huge
stone torii at the end of the village loomed ghostly against the
blackness of the forest. The glancing moonlight shot strange
shadows across the path, and we went whirling through this lattice of
light and darkness in stillness and solitude. The moon rose higher
and was hidden in the leafy arch overhead, and before we realized
that its faint light was fading, came flashes of lightning, the rumble of
approaching thunder, and a sudden crash, as the flood of rain struck
the tree-tops and poured through. The hoods of the jinrikishas were
drawn up, the oil-papers fastened across us, and through pitch
darkness the coolies raced along. Vivid flashes of lightning showed
the thick, white sheet of rain, which gusts of wind blew into our faces,
while insidious streams slipped down our shoulders and glided into
our laps. Putting their heads down, the coolies beat their way against
the rain for two more soaking miles to Imaichi, the last village on the
road, only five miles from Nikko. The tea-house into which we turned
for shelter was crowded with belated and storm-bound pilgrims
coming down from the sacred places of Nikko and Chiuzenji. All
Japanese are talkative, the lower in station the more loquacious, and
the whole coolie company was chattering at once. As the place was
too comfortless to stay in, we turned out again in the rain, and the
coolies splashed away at a walk, through a darkness so dense as to
be felt. At midnight our seven jinrikishas rattling into the hotel court,
and fourteen coolies shouting to one another as they unharnessed
and unpacked, roused the house and the whole neighborhood of
Nikko. Awakened sleepers up-stairs looked out at us and banged the
screens angrily, but no sounds can be deadened in a tea-house.
To the traveller the tea-house presents many phases of comfort,
interest, and amusement altogether wanting in the conventional
hotel, which is, unfortunately, becoming common on the great routes
of travel. The dimensions of every house in the empire conform to
certain unvarying rules. The verandas, or outer galleries of the
house, are always exactly three feet wide. A foreigner, who insisted
on a nine-feet-wide veranda, entailed upon his Nikko carpenter many
days of painful thought, pipe-smoking, and conference with his
fellows. These mechanics were utterly upset in their calculations.
They sawed the boards and beams too long or too short, and finally
produced a very bad, un-Japanese piece of work. The floors of these
galleries are polished to a wonderful smoothness and surface. They
are not varnished, nor oiled, nor waxed, but every morning rubbed
with a cloth wrung out of hot bath-water which contains oily matter
enough to give, in time, this peculiar lustre. Three years of daily
rubbing with a hot cloth are required to give a satisfactory result, and
every subsequent year adds to the richness of tone and polish, until
old tea-houses and temples disclose floors of common pine looking
like rosewood, or six-century-old oak.
The area of every room is some multiple of three feet, because the
soft tatami, or floor-mats, measure six feet in length by three in
width. These are woven of common straw and rushes, faced with a
closely-wrought mat of rice-straw. It is to save these tatami and the
polished floors that shoes are left outside the house.
The thick screens, ornamented with sketches or poems, that
separate one room from another, are the fusuma; the screens
shutting off the veranda, pretty lattice frames covered with rice-paper
that admit a peculiarly soft light to the rooms, are the shoji, and in
their management is involved an elaborate etiquette. In opening or
closing them, well-bred persons and trained servants kneel and use
each thumb and finger with ordered precision, while it is possible to
convey slight, contempt, and mortal insult in the manner of handling
these sliding doors. The outer veranda is closed at night and in bad
weather by amados, solid wooden screens or shutters, that rumble
and bang their way back and forth in their grooves. These amados
are without windows or air-holes, and the servants will not willingly
leave a gap for ventilation. “But thieves may get in, or the kappa!”
they cry, the kappa being a mythical animal always ready to fly away
with them. In every room is placed an andon, or night-lamp. If one
clap his hands at any hour of the twenty-four, he hears a chorus of
answering Hei! hei! hei’s! and the thump of the nesans’ bare feet, as
they run to attend him. While he talks to them, they keep ducking
and saying Heh! heh! which politely signifies that they are giving their
whole attention.
The Japanese bed is the floor, with a wooden box under the neck
for a pillow and a futon for a covering. To the foreigner the Japanese
landlord allows five or six futons, or cotton-wadded comforters, and
they make a tolerable mattress, although not springy, and rather apt
to be damp and musty. The traveller carries his own sheets, woolen
blankets, feather or air pillows, and flea-powder, the latter the most
necessary provision of all. The straw mats and the futons swarm with
fleas, and without a liberal powdering, or, better, an anointing with oil
of pennyroyal, it is impossible to sleep. These sleeping
arrangements are not really comfortable, and, after the fatigue of
walking and mountain-climbing, stiffen the joints. By day the futons
are placed in closets out of sight, or hung over the balconies to air,
coming back damper than ever, if the servants forget to bring them in
before sunset. The bedroom walls are the sliding paper screens; and
if one’s next neighbor be curious, he may slip the screen a little or
poke a hole through the paper. A whisper or a pin-drop travels from
room to room, and an Anglo-Saxon snorer would rock the whole
structure.
At ordinary Japanese inns the charge for a day’s accommodation
ranges from a half-yen upward. A Japanese can get his lodgings and
all his meals for about thirty cents, but foreigners are so clumsy,
untidy, and destructive, and they demand so many unusual things,
that they are charged the highest price, which includes lodging,
bedding, and all the tea, rice, and hot water they may wish. All other
things are extra. In the beaten tracks bread and fresh beef may
always be found, and each year there is less need of carrying the
supplies formerly so essential. Chairs and tables, cots, knives and
forks, and corkscrews have gradually penetrated to the remotest
mountain hamlets. At the so-called foreign hotels at Nikko and other
resorts, charges are usually made at a fixed price for each day, with
everything included, as at an American hotel.
Foreigners travelling away from the ports take with them a guide,
who acts as courier, cooks and serves the meals, and asks two and
a half yens a day with expenses. Thus accompanied, everything
goes smoothly and easily; rooms are found ready, meals are served
promptly, show-places open their doors, the best conveyances await
the traveller’s wish, and an encyclopædic interpreter is always at his
elbow. Without a guide or an experienced servant, even a resident
who speaks the language fares hardly. Like all Orientals, the
Japanese are impressed by a retinue and the appearances of
wealth. They wear their best clothes when travelling, make a great
show, and give liberal tips. The foreigner who goes to the
Nakasendo or to remote provinces alone, trusting to the phrase-
book, finds but little consideration or comfort. He ranks with the class
of pilgrims, and the guest-room and the choicest dishes are not for
him. The guide may swindle his master a little, but the comforts and
advantages he secures are well worth the cost. All the guides are
well-to-do men with tidy fortunes. They exact commissions wherever
they bring custom, and can make or break landlords or merchants if
they choose to combine. Some travellers, who, thinking it sharp to
deprive the guides of these percentages, have been left by them in
distant provinces and forced to make their way alone, have found the
rest of the journey a succession of impositions, difficulties, and even
of real hardships. After engaging a guide and handing him the
passport, the traveller has only to enjoy Japan and pay his bill at the
end of the journey. The guides know more than the guide-book; and
with Ito, made famous by Miss Bird, Nikko and Kioto yielded to us
many pleasures which we should otherwise have missed. An
acquaintance with Miyashta and his sweet-potato hash made the
Tokaido a straight and pleasant way; and Moto’s judicial
countenance caused Nikko, Chiuzenji, and Yumoto to disclose
unimagined beauties and luxuries; and Utaki always marshalled the
impossible and the unexpected.
CHAPTER XV
NIKKO
Of all Japan’s sacred places, Nikko, or Sun’s Brightness, is

dearest to the Japanese heart. Art, architecture, and landscape-
gardening add to Nature’s opulence, history and legend people it
with ancient splendors, and all the land is full of memories. “He who
has not seen Nikko cannot say Kekko!” (beautiful, splendid, superb),
runs the Japanese saying.
With its forest shades, its vast groves, and lofty avenues, its hush,
its calm religious air, Nikko is an ideal and dream-like place, where
rulers and prelates may well long to be buried, and where priests,
poets, scholars, artists, and pilgrims love to abide. Each day of a
whole summer has new charms, and Nikko’s strange fascination but
deepens with acquaintance.
The one long street of Hachi-ishi, or lower Nikko village, ends at
the banks of the Dayagawa, a roaring stream that courses down a
narrow valley, walled at its upper end by the bold, blue bar of
Nantaisan, the sacred mountain. Legend has peopled this valley of
the Dayagawa with impossible beings—giants, fairies, demons, and
monsters. Most of the national fairy stories begin with, “Once upon a
time in the Nikko mountains,” and one half expects to meet imp or
fay in the green shadows. Mound builder and prehistoric man had
lived their squalid lives here; the crudest and earliest forms of
religion had been observed in these forest sanctuaries long before
Kobo Daishi induced the Shinto priests to believe that their god of
the mountain was but a manifestation of Buddha. Everything
proclaims a hoary past—trees, moss-grown stones, battered images,
crumbling tombs, overgrown and forgotten graveyards.
Each summer half the Tokio legations move bodily to Nikko, and
temples, monastery wings, priests’ houses, and the homes of the
dwellers in the upper village are rented to foreigners in ever-
increasing numbers. Nikko habitations do not yet bring the prices of
Newport cottages, but the extravagant rate of three and even five
hundred yens for a season of three months is a Japanese
equivalent. Besides the foreigners, there are many Japanese
residents; and, while the Crown-Prince occupies his summer palace,
he is daily to be met in the streets, the forest paths, or temple
grounds. The white-clad pilgrims throng hither by thousands during
July and early August, march picturesquely to the jingle of their staffs
and bells round the great temples, and trudge on to the sanctuary on
Chiuzenji’s shores within the shadow of holy Nantaisan.
Two bridges cross the Daiyagawa, and lead to the groves and
temples that make Nikko’s fame. One bridge is an every-day affair of
plain, unpainted timbers, across which jinrikishas rumble noisily, and
figures pass and repass. The other is the sacred bridge, over which
only the Emperor may pass, in lieu of the Shoguns of old, for whom it
was reserved. It is built of wood, covered with red lacquer, with many
brass plates and tips, and rests on foundation piles of Titanic stone
columns, joined by cross-pieces of stone, carefully fitted and
mortised in. Tradition maintains that the gods sent down this rainbow
bridge from the clouds in answer to saintly prayer. Its sanctity is so
carefully preserved, that when the Emperor wished to pay the
highest conceivable honor to General Grant, he ordered the barrier
to the bridge to be opened that his guest might walk across. Greatly
to his credit, that modest soldier refused to accept this honor, lest it
should seem a desecration to the humble believers in the sanctity of
the red bridge.
Shaded avenues, broad staircases, and climbing slopes lead to
the gate-ways of the two great sanctuaries—the mortuary temples
and tombs of the Shogun Iyeyasu and his worthy grandson, the
Shogun Iyemitsu. The hill-side is shaded by magnificent old
cryptomerias; and these sacred groves, with the soft cathedral light
under the high canopy of leaves, are as wonderful as the sacred
buildings. Each splendid gate-way, as well as the soaring pagoda,
can be seen in fine perspective at the end of long avenues of trees,
and bronze or stone torii form lofty portals to the holy places. The
torii is a distinctively national structure, and these grand skeleton
gates of two columns and an upward curving cross-piece are
impressive and characteristic features of every Japanese landscape,
standing before even the tiniest shrines in the Liliputian gardens of
Japanese homes, as well as forming the approach to every temple.
The stone torii and the rows of stone lanterns are mossy and lichen-
covered, and every foot of terrace or embankment is spread with fine
velvety moss of the freshest green. Although two hundred years old,
the temples themselves are in as perfect condition and color as
when built; and nothing is finer, perhaps, than the five-storied
pagoda with its red lacquered walls, the brass trimmings of roofs and
rails, the discolored bells pendent from every angle, and a queer,
corkscrew spiral atop, the whole showing like a great piece of
jeweller’s work in a deep, green grove.
Iyeyasu, founder of Yeddo, successor of the Taiko, and military
ruler in the golden age of the arts in Japan, was the first Shogun
buried on Nikko’s sacred hill-side, and it was intended to make the
mortuary temple before his tomb as splendid as the crafts of the day
permitted. His grandson, Iyemitsu, was the next and only other
Shogun interred at Nikko, and his temple fairly rivals that of his
ancestor.
At each shrine rise broad stone steps leading to the first and outer
court-yards, where stand the magnificent gates, exquisitely carved,
set with superb metal plates, and all ablaze with color and gilding.
The eye is confused in the infinite detail of structure and ornament,
and the intricacy of beams and brackets upholding the heavy roofs of
these gate-ways. Walls of red lacquer and gold, with carved and
colored panels topped with black tiles, surround each enclosure, and
through inner and outer courts and gate-ways, growing ever more
and more splendid, the visitor approaches the temples proper, their
soaring roofs, curved gables, and ridge-poles set with the Tokugawa
crest in gold, sharp cut against the forest background. At the lowest
step his shoes are taken off, and he is permitted to wander slowly
through the magnificent buildings on the soft, silk-bordered mats.
Richly panelled ceilings, lacquered pillars, carved walls, and curtains
of the finest split bamboo belong to both alike, and in the gloom of
inner rooms are marvels of carving and decoration, only half visible.
Both temples were once splendid with all the emblems and
trappings of Buddhism, redolent with incense, musical with bells and
gongs, and resounding all day with chanted services. But after the
Restoration, when the Shinto became the state religion and the
Emperor made a pilgrimage to Nikko, Iyeyasu’s temple was stripped
of its splendid altar ornaments, banners, and symbols, and the
simple mirror and bits of paper of the empty Shinto creed were
substituted. In the dark chapel behind the first room there remains a
large gong, whose dark bowl rests on a silken pad, and when softly
struck fills the place with rising and falling, recurring and wavering,
tones of sweetness for five whole minutes, while Ito stands with open
watch and warning finger, and the priest bends low and drinks in the
music with ecstatic countenance. Iyemitsu’s temple was spared, and
there stand the rows of superb lacquered boxes containing the
sacred writings. There, too, are the gilded images, golden lotus-
leaves, massive candlesticks, drums, gongs, banners and pendent
ornaments, besides the giant koros, breathing forth pale clouds of
incense, that accompanied the rites of the grand old Buddhist faith.
INTERIOR OF THE IYEMITSU TEMPLE
Each temple has a fine water-tank in its outer court; an open

pavilion, with solid corner posts supporting the heavy and ornate roof
above the granite trough. Each basin is a single, huge block of
stone, hollowed out and cut with such exactness that the water,
welling up from the bottom, pours over the smooth edges so evenly
as to give it the look of a cube of polished glass. The fountain at the
Iyemitsu temple was the gift of the princes of Nabeshima, and its
eaves flutter with the myriad flags left there by pilgrims who come to
pray at the great shrine. All about the temple grounds is heard the
noise of rushing water, and the music and gurgle of these tiny
streams, the rustle of the high branches, and the cawing of huge
solitary rooks are the only sounds that break the stillness of the
enchanted groves between the soft boomings of the morning and
evening bells. The noise of voices is lost in the great leafy spaces,
and the sacredness of the place subdues even the unbelieving
foreigner, while native tourists and pilgrims move silently, or speak

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Handbook Of DNA Forensic

Applications And Interpretation 1st

Forensic Practitioner's Guide to the Interpretation of

Forensic DNA Applications: An Interdisciplinary

Applications of Nanovesicular Drug Delivery 1st Edition

Metamaterials for Antenna Applications 1st Edition Amit

Optimization Techniques and their Applications to Mine

Handbook of Metrology and Applications Dinesh K Aswal

Internet of Things Theory and Practice Build Smarter

Computational Analysis and Deep Learning for Medical

Handbook of DNA Forensic

ISBN 978-981-19-0042-6 ISBN 978-981-19-0043-3 (eBook)

© Springer Nature Singapore Pte Ltd. 2022

Part I Insights and Advancements in DNA Forensics

Part II Extraction and Analysis of DNA Samples in Laboratory

Part III DNA in Courtrooms

11 DNA-Based Human Identification in Mass-Disaster Cases . . . . . . . . . 149

Amit Kumar, Amita Kashyap, and Edwin Huffine

A. Kumar · A. Kashyap (B)

© Springer Nature Singapore Pte Ltd. 2022 3

The Master Molecule in Witness Box (with Some Case

DNA Forensic Investigation

Developing DNA Databases and Revolutionizing

Edwin Huffine, Amit Kumar, and Amita Kashyap

© Springer Nature Singapore Pte Ltd. 2022 11

Bioinformatics is the application of information technology in the storage, retrieval

Laboratory Information Management System is a software platform enabling effi-

Radio Frequency Identification system (RFID) is a means of tracking and identifi-

This technology relies on identification of an individual or object based on measure-

for uniqueness of an individual is SNPs single nucleotide polymorphisms yielding a

AI and IoT in Forensic Investigations

Technical Challenges to DNA Profiling and Forensic Analysis

Challenging, Degraded and Unusual Samples

Touch DNA/Trace DNA

DNA from Harsh Environmental Samples

Obtaining DNA from Challenging Sources

Fig. 2.3 Steps in pre amplification

© Springer Nature Singapore Pte Ltd. 2022 25

testing could lead to produce thousands of identifications, a considerable amount of

Amit Kumar and Amita Kashyap

DNA Sample Identification and Its Integrity in Crime Scene

Any biological or non-biological elements that are expected to be useful in analyzing

Contamination and Elimination Sample

A. Kumar (B) · A. Kashyap

© Springer Nature Singapore Pte Ltd. 2022 35

Sources to Obtain DNA from Crime Scene

Sample Collection for DNA Profiling

Fig. 4.1 DNA evidence

Of all Japan’s sacred places, Nikko, or Sun’s Brightness, is

Each temple has a fine water-tank in its outer court; an open

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