DNA PROFILING

ASSIGNMENT # 2 SEMESTER SPRING-2021

Submission Date (March 23, 2021)

BY

AREEJ ANWAR

ROLL # 17161624-046

LAW-407 (Evidence Law)

LL.B. (Bachelors of Laws) CAM

Submitted To

SIR NASIR MAJEED

School of Law

UNIVERSITY OF GUJRAT
DNA fingerprinting is a laboratory technique used to establish a link between biological
evidence and a suspect in a criminal investigation. A DNA sample taken from a crime scene is
compared with a DNA sample from a suspect. If the two DNA profiles are a match, then the
evidence came from that suspect. Conversely, if the two DNA profiles do not match, then the
evidence cannot have come from the suspect. DNA fingerprinting is also used to establish
paternity.
DNA fingerprinting, also called DNA typing, DNA profiling, genetic
fingerprinting, genotyping, or identity testing, in genetics, method of isolating and identifying
variable elements within the base-pair sequence of DNA (deoxyribonucleic acid). The technique
was developed in 1984 by British geneticist Alec Jeffreys, after he noticed that certain
sequences of highly variable DNA (known as minisatellites), which do not contribute to the
functions of genes, are repeated within genes. Jeffreys recognized that each individual has a
unique pattern of minisatellites (the only exceptions being multiple individuals from a
single zygote, such as identical twins)

DIFFERENT DNA PROFILING METHODS

Restriction Fragment Length Polymorphism (RFLP) Technique
Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the
English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis
of unique patterns in DNA fragments in order to genetically differentiate between organisms –
these patterns are called Variable Number of Tandem Repeats (VNTRs).
Genetic polymorphism is defined as the inherited genetic differences among individuals in over
1% of normal population. The RFLP technique exploits these differences in DNA sequences to
recognize and study both intraspecies and interspecies variation.
Principle
Restriction endonucleases are enzymes that cut lengthy DNA into short pieces. Each restriction
endonuclease targets different nucleotide sequences in a DNA strand and therefore cuts at
different sites.
The distance between the cleavage sites of a certain restriction endonuclease differs between
individuals. Hence, the length of the DNA fragments produced by a restriction endonuclease
will differ across both individual organisms and species.
How does it Work?
RFLP is performed using a series of steps briefly outlined below:
DNA Extraction
To begin with, DNA is extracted from blood, saliva or other samples and purified.
DNA Fragmentation
The purified DNA is digested using restriction endonucleases. The recognition sites of these
enzymes are generally 4 to 6 base pairs in length. The shorter the sequence recognized, the
greater the number of fragments generated from digestion.
For example, if there is a short sequence of GAGC that occurs repeatedly in a sample of DNA.
The restriction endonuclease that recognizes the GAGC sequence cuts the DNA at every
repetition of the GAGC pattern.
If one sample repeats the GAGC sequence 4 times whilst another sample repeats it 2 times, the
length of the fragments generated by the enzyme for the two samples will be different.
Gel Electrophoresis
The restriction fragments produced during DNA fragmentation are analyzed using gel
electrophoresis.
The fragments are negatively charged and can be easily separated by electrophoresis, which
separates molecules based on their size and charge. The fragmented DNA samples are placed in
the chamber containing the electrophoretic gel and two electrodes.
When an electric field is applied, the fragments migrate towards the positive electrode. Smaller
fragments move faster through the gel leaving the larger ones behind and thus the DNA samples
are separated into distinct bands on the gel.
Visualization of Bands
The gel is treated with luminescent dyes in order to make the DNA bands visible.
Applications of RFLP
RFLP has been used for several genetic analysis applications since its invention.
Some of these key applications of RFLP are listed below:
 To determine the status of genetic diseases such as Cystic Fibrosis in an individual.
 To determine or confirm the source of a DNA sample such as in paternity tests or
criminal investigations.
 In genetic mapping to determine recombination rates that show the genetic distance
between the loci.
 To identify a carrier of a disease-causing mutation in a family.
Disadvantages of RFLP
Since its invention, RFLP has been  a widely used genome analysis techniques employed in
forensic science, medicine, and genetic studies. However, it has become almost obsolete with
the advent of relatively simple and less expensive DNA profiling technologies such as the
polymerase chain reaction (PCR).
The RFLP procedure requires numerous steps and takes weeks to yield results, while techniques
such as PCR can amplify target DNA sequences in a mere few hours.
Additionally, RFLP requires a large DNA sample, the isolation of which can be a laborious and
time-consuming process. In contrast, PCR can amplify minute amounts of DNA in a matter of
hours.
Due to numerous reasons such as these, the PCR technique has largely replaced RFLP in most
applications requiring DNA sequencing such as paternity testing or forensic sample analysis.
Furthermore, the identification of single-nucleotide polymorphisms in the Human Genome
Project has almost replaced the need for RFLP in disease status analysis
POLYMERASE CHAIN REACTION ANALYSIS
 Polymerase chain reaction, or PCR, is a technique to make many copies of a specific
DNA region in vitro (in a test tube rather than an organism).
 PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires
DNA primers designed specifically for the DNA region of interest.
 In PCR, the reaction is repeatedly cycled through a series of temperature changes, which
allow many copies of the target region to be produced.
 PCR has many research and practical applications. It is routinely used in DNA cloning,
medical diagnostics, and forensic analysis of DNA
Developed by Kary Mullis in 1983, a process was reported by which specific portions of the sample
DNA can be amplified almost indefinitely1. The process, polymerase chain reaction (PCR), mimics
the biological process of DNA replication, but confines it to specific DNA sequences of interest. With
the invention of the PCR technique, DNA profiling took huge strides forward in both discriminating
power and the ability to recover information from very small (or degraded) starting samples.
PCR greatly amplifies the amounts of a specific region of DNA. In the PCR process, the DNA
sample is denatured into the separate individual polynucleotide strands through heating.
Two oligonucleotide DNA primers are used to hybridize to two corresponding nearby sites on
opposite DNA strands in such a fashion that the normal enzymatic extension of the active terminal
of each primer (that is, the 3’ end) leads toward the other primer. PCR uses replication enzymes
that are tolerant of high temperatures, such as the thermostable Taq polymerase. In this fashion,
two new copies of the sequence of interest are generated. Repeated denaturation, hybridization,
and extension in this fashion produce an exponentially growing number of copies of the DNA of
interest. Instruments that perform thermal cycling are readily available from commercial sources.
This process can produce a million-fold or greater amplification of the desired region in 2 hours or
less.

1
(Saiki et al. 1985, 1985)
Using gel electrophoresis to visualize the results of PCR
The results of a PCR reaction are usually visualized (made visible) using gel
electrophoresis. Gel electrophoresis is a technique in which fragments of DNA are pulled
through a gel matrix by an electric current, and it separates DNA fragments according to size. A
standard, or DNA ladder, is typically included so that the size of the fragments in the PCR
sample can be determined.
DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel
is stained with a DNA-binding dye. For example, a PCR reaction producing a 400400400 base
pair (bp) fragment would look like this on a gel:
A DNA band contains many, many copies of the target DNA region, not just one or a few
copies. Because DNA is microscopic, lots of copies of it must be present before we can see it by
eye. This is a big part of why PCR is an important tool: it produces enough copies of a DNA
sequence that we can see or manipulate that region of DNA.
STR analysis
STRs are repetitive sequence elements 3–7 base pairs in length scattered throughout the human
genome. By amplifying and analyzing these polymorphic loci, and then comparing the resulting
STR profile to that of a reference sample, the origin of biological samples such as cells or tissues
can be identified and verified. The more loci that are amplified, the higher the statistical power
of discrimination. For example, when analyzing the 15 STR loci amplified by the PowerPlex®
16 HS System, the power of discrimination is as high as 1 in 1.42 × 1018, making it highly
unlikely that two DNA profiles will match at random.
The system of DNA profiling used today is based on polymerase chain reaction (PCR) and uses
simple sequences[ or short tandem repeats (STR). This method uses highly polymorphic regions
that have short repeated sequences of DNA (the most common is 4 bases repeated, but there are
other lengths in use, including 3 and 5 bases). Because unrelated people almost certainly have
different numbers of repeat units, STRs can be used to discriminate between unrelated
individuals. These STR loci (locations on a chromosome) are targeted with sequence-specific
primers and amplified using PCR. The DNA fragments that result are then separated and
detected using electrophoresis. There are two common methods of separation and
detection, capillary electrophoresis (CE) and gel electrophoresis.
Each STR is polymorphic, but the number of alleles is very small. Typically, each STR allele
will be shared by around 5–20% of individuals. The power of STR analysis derives from
inspecting multiple STR loci simultaneously. The pattern of alleles can identify an individual
quite accurately. Thus STR analysis provides an excellent identification tool. The more STR
regions that are tested in an individual the more discriminating the test becomes.
Mitochondrial analysis
For highly degraded samples, it is sometimes impossible to get a complete profile of the 13
CODIS STRs. In these situations, mitochondrial DNA (mtDNA) is sometimes typed due to there
being many copies of mtDNA in a cell, while there may only be 1–2 copies of the nuclear DNA.
Forensic scientists amplify the HV1 and HV2 regions of the mtDNA, and then sequence each
region and compare single-nucleotide differences to a reference. Because mtDNA is maternally
inherited, directly linked maternal relatives can be used as match references, such as one's
maternal grandmother's daughter's son. In general, a difference of two or more nucleotides is
considered to be an exclusion. Heteroplasmy and poly-C differences may throw off straight
sequence comparisons, so some expertise on the part of the analyst is required. mtDNA is useful
in determining clear identities, such as those of missing people when a maternally linked relative
can be found. in determining clear identities, such as those of missing people when a maternally
linked relative can be found.
mtDNA can be obtained from such material as hair shafts and old bones/teeth. Control
mechanism based on interaction point with data. This can be determined by tooled placement in
sample.
DECONVOLUTION OF MIXED BLOOD
1. Separation of white cells and DNA typing Selective collection of human blood cells was
performed by using three cuttings of the stain (1 cm2 ) in three microcentrifuge tubes
labelled as A, B and O. 1 ml saline was added in the tubes, vortexed and incubated for 1
hour at 37°C. Anti-A, anti-B and anti-H lectin were added to the respective tubes (A, B
and O) and incubated for 10 minutes at 37°C. These microcentrifuge tubes were then
centrifuged at 500 g for 5 minutes and the supernatant was separated. The pellet obtained
was dissolved in 30 µl TE buffer and preserved at 4°C. A drop of sample was then
placed on FTA™ paper, dried and processed for DNA extraction.
2. Messenger RNA (mRNA) biomarkers have been employed to identify the origin of body
fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be
applied to identify individuals in mixture samples composed of two body fluids. In this
study, we selected five blood-specific mRNA biomarkers of venous blood (SPTB,
CD3G, AMICA1, ANK1, and GYPA) that encompass 16 SNPs to identify the mixture
contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva, and
 vaginal secretions were amplified and typed as body-fluid positive controls. We
established the system of multiplex PCR and single base extension (SBE) reaction
followed by CE. The amplicon size was between 90bp and 294bp. The peripheral blood
specificity was examined against other human body fluids, including saliva, semen, skin,
menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and
could be successfully typed in homemade mixtures which are composed of different
body fluids with 1 ng peripheral blood mRNA added. This system showed a
supersensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other
body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese
population. It was the first time to establish a method for identifying the blood donors
and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot
assay. This peripheral blood specific SNP typing system showed high sensitivity to the
typing of blood source specific markers regardless of other body fluids in the mixture.
Use of centrifuge
A dose of whole donor blood is placed in a large centrifuge and is spun for a preset time
(usually about 15 minutes) at a preset speed. The red blood cells precipitate to the bottom of
the bag, with the platelets above them, then the white blood cells and the plasma at the very
top.
Centrifugal force is used to separate the components of blood – red blood cells, platelets and
plasma – from each other. The result is that the particles with different densities precipitate
in layers.
DNA FROM BLOOD
Whole blood DNA isolation using magnetic beads works by capturing DNA on magnetic beads
coated with a matrix of silica for binding nucleic acids. As with the precipitation chemistry
methods, the whole blood cells first must be lysed using SDS or similar detergents.