DNA MOLECULAR MARKERS

FACILITATE GENETIC
PARENTAGE ANALYSIS
INDRIAWATI, M.Si
MOLECULAR DNA PRACTICIST
JAKARTA
 • Basis of DNA Profiling
 The genome of each individual is unique (with the exception of identical
twins) and is inherited from parents

 Probe subsets of genetic variation in order to differentiate between

individuals (statistical probabilities of a random match are used)

 DNA typing must be performed efficiently and reproducibly (information

must hold up in court)

 Current standard DNA tests DO NOT look at genes – little/no information

about race, predisposal to disease, or phenotypical information (eye color,
height, hair color) is obtained
• DNA in the Cell
 • Glossary
 Fenotip adalah sifat penampilan fisik yang bisa diamati, seperti warna, bentuk, dan tinggi
 Genotip adalah jenis gen yang mengendalikan fenotip

 Homozigot adalah pewarisan alel identik dari kedua orang tua

biologis
Contoh:
Ketika ayah dan ibu sama-sama menurunkan alel mata cokelat
dominan (CC) ke anaknya, sehingga ia bermata cokelat (CC)

 Heterozigot adalah pewarisan dua alel berbeda dan ditandai

dengan salah satu alel bersifat dominan sementara lainnya resesif.

Contoh:

ibu mancung tetapi anaknya tidak mancung

Pada tumbuhan tinggi heterozigot, satu alel T dan satu alel t
menunjukkan tinggi. T adalah yang dominan dan t adalah yang
resesif. Tt mewakili heterozigot.
 • Genes, Chromosomes, DNA

histone: Protein associated with a chromosome; aids in

chromosome formation.

sex chromosome: X or Y chromosome (in humans).

SRY: Sex-determining region Y gene; gene that triggers an

embryo to develop into a male.
A DNA paternity test is considered the gold
standard by both the scientific and legal
communities when it comes to accurately
establishing a relationship between a
possible father and a child.
Application for DNA Testing
Crime solving – matching suspect with evidence…
Accident victims – after airplane crashes…
Soldiers in war – who is the “unknown” soldier…
Paternity testing – who is the father…
Immigration testing – are two people related…
Missing persons investigations – whose remains…
Source of DNA for parentage Checking

1. Blood
2. Semen
3. Saliva
4. Urine
5. Hair
6. Teeth
7. Bone
8. Tissue
9. etc.
DNA ISOLATION
 DNA ISOLATION (2)
• Ekstraksi
 DNA Isolation (3)
• Presipitasi
DNA TEST METHOD

1. Restriction Fragment Length Polymorphism (RFLP) –

Non PCR
2. PCR Analysis
• Mitochondrial DNA Analysis
• Y-Chromosome Analysis
• Mt-DNA FEMALES
• PATH OF Y-DNA MALES
• RFLP METHOD

• Requires larger amounts of DNA,

RFLP testing requires a relatively
large amount of DNA (~50ng =
thousands of cells)
• Not ideal for forensic evidence,
in which small, degraded
samples are common
 • PCR METHOD
• Polymerase Chain Reaction = molecular Xeroxing
• Three temperature phases, carried out in a Thermal Cycler,
replicate or “amplify” the desired DNA fragment(s)

Dr. Kary Mullis

Eccentric Genius

Denaturasi annealing primer Replikasi DNA

Target Amplifikasi : Siklus PCR diulang

No. of No. Amplicon Cycles

1 cycle = 2 Amplicon
Copies of Target
2 cycle = 4 Amplicon
1 2
3 cycle = 8 Amplicon

2 4
4 cycle = 16 Amplicon
3 8
5 cycle = 32 Amplicon
4 16

6 cycle = 64 Amplicon 5 32

6 64

7 cycle = 128 Amplicon 20 1,048,576

30 1,073,741,824
• PCR PROCESS

Target Sequence

Target Sequence

PROSES PCR : Amplifikasi

1 – Denaturasi oleh panas
2 – Annealing
Penempelan pasangan primer
berlabel biotin di kedua ujung Sekuen Target

ce
Target Sequen
3’

5’
5’
3’ Biotin
Primer 1
Primer 2
Biotin
3’
5’
5’

3’
ce
Target Sequen
3 – Ekstension
Taq DNA Polymerase mengkatalisa pemanjangan Primer
sebagai Komplemen Nukleotida

ce
Target Sequen
3’

5’
5’
3’
Biotin
Primer 1
Taq DNA
Polymerase
Primer 2
Biotin 3’
5’
5’

3’
ce
Target Sequen
Akhir Siklus PCR ke-1
Hasil 2 tiruan dari Sekuen Target

ce
Target Sequen

Biotin

Biotin

ce
Target Sequen
Persyaratan PCR:

1. Genom DNA  Hasil Isolasi DNA dari darah, jaringan,

tulang, dsb

2. Primer/Markers Sekuen DNA penanda unik terdiri

dari 20-30basa yang mengapit DNA target, sebagai titik
awal sintesis DNA
• PCR COMPONENT
DNA e.g. purified from blood
Enzyme (Taq– Polymerase) Enzyme is capable of copying DNA by
producing complementary DNA strands
starting from a primer bound to a single-
stranded template.
2 PCR Primers Short pieces of single-stranded DNA;
specific for a certain target DNA sequence,
used as a starting point of DNA polymerase
activity.
Nucleotides Smallest Unit of DNA; “letters“ of the
biological alphabet
Buffer Conditions Reaction buffer ( Salt....)
• MARKERS/PRIMER/PENANDA (1)
• MARKERS/PRIMER/PENANDA (2)
Can be used for tiny
samples or degraded
samples because it makes
many copies of sample.
• DNA AMPLIFIKASI
 Polymerase Chain Reaction-
PCR
 Amount of starting DNA is
increased exponentially with
each cycle
• SHORT TANDEM REPEAT PROCEDURE
PATERNITY
CASES
• Y CHROMOSOME
STR PROFILING
Short Tandem Repeats (STR)
• Basis of forensic DNA testing
• Short stretches of DNA characterized by a repeat unit
• Repeats present in variable numbers in individuals
• Number of repeats distinguishes one person from another
• Forensic DNA analysis designed to isolate, count and compare
STR’s between individuals

maternal
chromosome
paternal
chromosome
 Short Tandem Repeats (STRs)
AATG

~ ~
7 repeats
~ ~
8 repeats
 Repeat region is variable (polymorphic)
 Each variant is referred to as an allele
 Flanking region is constant
KEY: Alleles are distinguished by length

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another
STR Analysis
STR Analysis

 Genotyping is performed by
comparing to STR allelic ladder
STR allelic ladder represents all
possible STR designations for a given
DNA site
 Alleles represent different lengths of
STRs on a chromosome
 Sizing assured by internal sizing
standard
“Specialized” PCR-based systems

•mtDNA
•Y-STRs
•SNPs
Other Applications of DNA Analysis

IDENTIFICATION OF MASS DISASTER VICTIMS

 World Trade Center, Tsunami, Hurricane Katrina
 Comparison of biological samples from the scene of disaster (bone,
teeth, hair) to personal effects from a missing person (razor, toothbrush)
Mitochondrial DNA
(mtDNA)
 Mitochondrial DNA (mtDNA)
Pros
• Single-cell sensitivity because each
cell contains ~1000 mitochondria
• Especially useful for shed hairs, burnt
remains
• Can be used to establish kinship
directly because entire complement
of mtDNA is maternally inherited
Cons
• Single-cell sensitivity because each cell
contains ~1000 mitochondria = very high
contamination risk!
• Heteroplasmy - more than one mtDNA
type manifesting in different tissues in the
same individual
• Lower power of discrimination - maternal
relatives all share the same mtDNA
 Y-STRs
Problem: Solution:
• ~99% of violent crimes • Test for
are committed by men
markers
• DNA Mixtures of male found only
suspect and female victim
can pose an analytical
on the Y-
challenge, especially chromosome
when the female  Only male
contribution is much DNA is
greater than the male = amplified!
preferential amplification
Single Nucleotide Polymorphisms (SNPs)

• Point mutations (base substitutions) found in 1% or more

of the population
• 1.8 million identified in human genome
• Detected on micro-array plates with fluorescent tags (all
or nothing response)
• ~50 SNPs provides same power of discrimination as 13 STR
loci
• Certain SNPs used as predictors of ancestry/ethnicity by a
private sector lab (DNA Witness)
FAMILY RELATIONSHIPS