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FORENSIC

GENETICS

FORENSIC GENETICS

• PERSONAL IDENTIFICATION

• KINSHIP ANALYSIS

• Paternity testing, even after death

• Establishing human corpse identity

• Putting together tissues coming from particular

persons in mass disasters

• Missing persons investigations

FORENSIC GENETICS

• Crime cases – matching suspect with evidence

• creating population data frequency

used for evaluation of proof value of case report

• establishing databases of convicted felons

used for linking unsolved crime cases with serial

offenders

• establishing databases of missing persons

used for identification of unknown human remains

FORENSIC GENETICS

Sources of biological evidence

• Blood

• Semen

• Saliva

• Urine

• Hair

• Teeth

• Bone

• Tissue

Somatic cell

Hair root 3-6 pg DNA DNA profileSperm cell (0.000000000003 g DNA)

DNA in the cell

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Cells without nucleus

Mature red blood cells, hair shaft

DNA in the Cellchromosome

cell nucleus

double stranded DNA molecule

Individual nucleotides

A T G C

HUMAN GENOME = 3 bilion base pairsPrinciples of Mendelian inheritance

The law of segregation

Each individual has two alleles lying oppositeto each other in the pair of homological chromosomes,

one inherit from the mother and second inheritfrom the father

The law of independent assortment

The pair of alleles coding one marker is independent

of the pair of alleles coding another marker

DNA POLIMORPHISM

VNTR - Variable number of tandem repeat

Sequence polymorphism

SNP - Single nucleotide polymorphism

Lenght polymorphism

Variable number of tandem repeat (VNTR)

The number of repeats can differ between individuals

10-100 nucleotides = minisatellites

AATGGCTCTTATGACGTATCATGACTAG

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Variable number of tandem repeat (VNTR)

The number of repeats can differ between individuals

AATG AATGAATG

2-6 nucleotides = microsatellites = STR (short tandem repeat)

Variable number of tandem repeat (STR)

9 repeats

AATG AATGAATG

5 repeats

locus allele 5,9 genotype 5/9

Heterozygote = the genotype at the locus has two different alleles

Variable number of tandem repeat (STR)

locus alleles 7,7 genotype 7/7

Homozygote = the genotype at locus has two copies of the same allele

AATG AATGAATG

7 repeats

7 repeats

Schema of a gel electrophoresis system

DNA detection in capillary sequencer

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MPS – massive parallel sequencing VNTR analysis in paternity testing

1-allaged father 2- child 3-mother M –height marker

1 2 3 M1 2 3 M

STR analysis in paternity testing

Without exclusion

Paternity exclusion

Paternity exclusion principlesArising from Mendelian inheritance rules

New feature is present in the child, while it is absent in the alleged father

and the mother

The child does not inherit any feature

from the alleged father

and or

PCR – Polymerase chain reaction

Enables to produce millions of copies

of a specific DNA sequence in approximately two hours

Classical PCR reaction mix contains template DNA, polymerase,

deoxynucleotides and a pair of primers – forward primer and reverse one

Multiplex PCR reaction mix contains

multiple primer pairs

even more than 20 primer pairs

Particular primer pairs are labeled

with different fluorescent dyes

Multiplex PCR

Multiple STR regions are examined simultaneously

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Advantages of Multiplex PCR

Anables analysis of degraded DNA

Makes more effective analysis of mixtures

Reduces template required

Reduces labour to obtain results

Increases the informativeness of the DNA test

STR Genotyping after fluorescent detection

Y- CHROMOSOME MARKERSspecific only for man

Haplotype Y - Genetic variation at multiple points along the Y chromosome

Y-STR haplotyping after fluorescent detection

W h y t h e Y - C h r o m o s o m e ?

98% of violent crimes is caused by men

Applications

– identificaton of male or male components in mixture

– istablishing paternity for male offspring

– paternal lineages testing

– male gender identification

Male – specific Y- STR markers

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GENDER IDENTIFICATION

WITH

AMELOGENIN

AMG Y (112 bp)

AMG X (106 bp)

DNA POLIMORPHISM

VNTR - Variable number of tandem repeat

Sequence polymorphism

SNP - Single nucleotide polymorphism

Lenght polymorphism

We differ from each other in 1 bp out of every 300 nucleobases

Single nucleotide polymorphism (SNP)

HomozygoteHomozygote

Heterozygote

Single nucleotide polymorphism (SNP)

G GA A

G A

Advantage of SNP over VNTR markers

� are abundant and common in the human genome

� have low mutation rate

� due to the small molecular weight they are very useful inanalysis of high degraded DNA samples

15 STR = 50 SNP0,2 ng 0.02 ng

Polymorphism of mtDNA

HV1 HV2

16365

0

73

16024 340

16 569 bp

HV1, HV2 – variable control70 regions type of SNP

ANDERSON SEQUENCE

Extractions of DNALimited & Old & Degraded

samples as in tissues such as

- bones & teeth- skeletal remains

- hair shafts !

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NON RECOMBINATON MARKERS

HAPLOID MARKERS

Mitochondrial DNA Y-chromosome DNA

Uninparental inheritance Inheritance from only one parent

from mother to her child from father to his son

PASSED DOWN

LINEAGES MARKERS

DNAExtraction

PCR amplifiationmultiple STR/SNP

markers

Separation and detection of

DNA alleles

Evaluation of DNAquantity and quality

Steps involved in processing forensic DNA samples

FORENSIC DNA EVIDENCEINTERPRETATION

Kinship Analysis

paternity testing

It is the percentage of the unjustly sued man who will be excluded as fathers in the course of investigation

PE determines usefulness for paternity testing

The more polymorphic the investigated markers are, the easier it is to exclude paternity of the man using these markers

PE - POWER OF EXCLUSIONPE for first degree expertise as well as DNA expertise

80%

85%

90%

95%

100%

ABO PGM

Kell ACP

MN GLO

Rh ESD

GM HP

I º EXPERTISE PE=84%

STR-Multiplex

15 loci

DNA EXPERTISE PE=99,9999%

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AN OPINION IN PATERNITY TESTING

I º E X P E R T I S E

EXCLUSION

RECONSTRUCTION OF GENOTYPE OF DEAD PERSON

LACK OF EXCLUSION ?

AN OPINION IN PATERNITY TESTING

D N A E X P E R T I S E

EXCLUSION with 4 ( four )

INDEPENDENT MARKERS

RECONSTRUCTION OF GENOTYPE OF DEAD PERSON

International Society of Forensic GeneticPolish Society of Forensic Medicine and Cryminology

CONFIRMATION

probability of patrernity = 99.9999 %

Paternity

Index (PI)

Probability of

Paternity

10

1001000

10 000

100 0001 000 000

90 %

99 %99.9 %99.99 %

99.999 %99.9999 %

Paternity testing evidence

Requirements for issuing a report witha positive weight for paternity

FORENSIC DNA EVIDENCEINTERPRETATION

personal identification

Probability that two unrelated individuals will have

different sets of genetic alleles

PD determines usefulness for personal identification

The more polymorphic markers are examined, the greater the chance that two unrelated individuals will not have an identical set

of these markers

PD - POWER OF DISCRIMINATIONPD IN PERSONAL IDENTIFICATION

ABORh

KELLMN

GMESDACP

PGMGLO

HP

PD ≈ 95 %

D16S539D3S1358

D2S1338D8S1179

D18S51D12S11D19S433

vWATH01

FGA

PD = 99.9999999 %

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Comparative analysis of received DNA profile

If match occurs, calculation of DNA profile

frequency

GENETIC IDENTIFICATION

No matchExclusion

MatchInclusion

Generation of case reportwith random match

probability

CALCULATION OF PROFILE FREQUENCY

AA = a2

Genotypes frequency BB = b2

AB = 2ab

taking into account assumption of HWE

If allele in locus A has frequency aand allele in locus B has frequency b

EVIDENCE VALUE OF PERSONAL IDENTIFICATION

Product of multiplication genotypes frequency

= Profile frequency PF

����

PF = GF1 marker x GF2 marker x GF3 marker x .......

1 = 1 PROFILE in .... persons

PROFILE FREQUENCY

The chance that a randomly selected individual from

a population will have an identical DNA genotype

such as another individual in the population

RANDOM MATCH PROBABILITY

RANDOM MATCH PROBABILITY USING 13 LOCI

DNA MOLECULAR POLYMORPHISM DACTYLOSCOPY