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A Primer on DNA Analysis and Human A Primer on DNA Analysis and Human Genetics, Applications for the Identification of Genetics, Applications for the Identification of 

Missing Persons and Human RemainsMissing Persons and Human RemainsMissing Persons and Human RemainsMissing Persons and Human Remains

Arthur J. Eisenberg, PhDProfessor Department of Pathology and Human Identification

Co‐Director UNT Center for Human IdentificationUniversity of North Texas Health Science Center

Fort Worth, Texas USA

DNA Testing for Human Identification

• Paternity testing ‐‐ identifying biological father• Immigration testing• Forensic cases ‐‐matching suspect with evidence

• Convicted felon DNA databases• Unidentified Human Remains• Missing persons investigations• Mass disasters ‐‐ putting pieces back together

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Sources of DNA from Biological Samples

BloodSemenSaliva (Buccal Swabs)Saliva (Buccal Swabs)UrineUrineHairTeethTeethBoneBoneTissueTissue

The Science of DNA for Human Identification

DNA is Responsible for Transmitting Hereditary Characteristics

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Human Cells Contain Two Kinds of DNA in Their Nucleus

Nuclear DNA and Mitochondrial DNANuclear DNA and Mitochondrial DNA

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The Nucleus Contains 23 Pairs of Chromosomes

Genetic Variation

DNA sequences at a particular chromosomal location that can h diff t l th d thave many different lengths due toVariation in the Number of TandemRepeats of a short DNA sequence

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Alleles – Contain Varying Numbers of 4 Base Pair Repeats

5 A A T G

A lle le :

A A T G A A T G A A T G A A T G

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A A T G A A T G A A T G A A T G A A T G

A A T G A A T G A A T G A A T G A A T G

A A T G

A A T G A A T G

F la n k in g re g io n o f u n iq u e s e q u e n c e

DNA in the Cell

Target Region for PCR

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Forensic DNA Testing Analyzes  Regions Containing Short Tandem Repeats (STR’s)

S H O R T T A N D E M R E P E A T S :T e t r a m e r s

S H O R T T A N D E M R E P E A T S :T e t r a m e r s

A G A TA G A T A G A TA G A T A G A TA G A T A G A TA G A T

A A A GA A A G A A A GA A A G A A A GA A A G A A A GA A A G

A A T GA A T G A A T GA A T G A A T GA A T G A A T GA A T G

A A A TA A A T A A A TA A A T A A A TA A A T A A A TA A A T

Starting DNA

Template

5’

5’

3’

3’

5’

5’3’

3’

DNA Amplification with the Polymerase Chain Reaction (PCR)

Make copies (extend primers)

5’

5’

3’

3’

Add primers (anneal) 5’3’

3’5’Forward primer

Reverse primer

Separate strands

(denature)

PCR Copies DNA Exponentially through Multiple Thermal Cycles

Original DNA target region

Thermal cycle

In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

Thermal cycle

Thermal cycle

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Short Tandem Repeats (STRs)

7 repeats

8 repeats

AATGAATG 1 repeat

The repeat region is variable between samples while the flanking regions where PCR primers bind are constant

p

Homozygote =  both alleles are the same lengthHeterozygote = alleles differ in length and can be 

resolved from one another

13 CODIS Core STR Loci

CSF1PO

D5S818TH01

TPOX

D7S820

D8S1179

D3S1358

FGAVWA

CSF1PO

D21S11

D13S317

D16S539D18S51

AMEL

AMEL

LociLoci Chromosomal Chromosomal LocationLocation

Genomic Genomic LocationLocationaa

Repeat MotifRepeat Motif Allele Size Allele Size RangeRange

Number of Number of segregating segregating alleles (k)alleles (k)bb

Possible Possible number of number of genotypes, genotypes,

k(k+1)/2k(k+1)/2

CSF1POCSF1PO 5q33.15q33.1 Ch.5; Ch.5; 149.484Mb149.484Mb

TAGATAGA 55--1616 2020 210210

FGAFGA 4q31.34q31.3 Ch.4; Ch.4; 156.086Mb156.086Mb

CTTTCTTT 12.212.2--51.251.2 8080 3,2403,240

THO1THO1 11p15.511p15.5 Ch.11; Ch.11; 2.156Mb2.156Mb

TCATTCAT 33--1414 2020 210210

TPOXTPOX 2p25.32p25.3 Ch.2; Ch.2; 1 436Mb1 436Mb

GAATGAAT 44--1616 1515 120120

Characteristics of the 13 Core CODIS STR Loci 

1.436Mb1.436MbvWAvWA 12p13.3112p13.31 Ch.12; Ch.12;

19.826Mb19.826Mb[TCTG][TCTA][TCTG][TCTA] 1010--2525 2828 406406

D3S1358D3S1358 3p21.313p21.31 Ch.3; Ch.3; 45.543Mb45.543Mb

[TCTG][TCTA][TCTG][TCTA] 88--2121 2424 300300

D5S818D5S818 5q23,25q23,2 Ch.5; Ch.5; 123.187Mb123.187Mb

AGATAGAT 77--1818 1515 120120

D7S820D7S820 7q21.117q21.11 Ch.7; Ch.7; 83.401Mb83.401Mb

GATAGATA 55--1616 3030 465465

D8S1179D8S1179 8q24.138q24.13 Ch.8; Ch.8; 125.863Mb125.863Mb

[TCTA][TCTG][TCTA][TCTG] 77--2020 1717 153153

D13S317D13S317 13q31.113q31.1 Ch.13; Ch.13; 80.52Mb80.52Mb

TATCTATC 55--1616 1717 153153

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D16S539D16S539 16q24.116q24.1 Ch.16; Ch.16; 86.168Mb86.168Mb

GATAGATA 55--1616 1919 190190

D18S51D18S51 18q21.3318q21.33 Ch.18; Ch.18; 59.098Mb59.098Mb

AGAAAGAA 77--39.239.2 5151 1,3261,326

Possible Possible number of number of genotypes, genotypes,

k(k+1)/2k(k+1)/2

Number of Number of segregating segregating alleles (k)alleles (k)bb

Allele Size Allele Size RangeRange

Repeat MotifRepeat MotifGenomic Genomic LocationLocationaa

Chromosomal Chromosomal LocationLocation

LociLoci

Characteristics of the 13 Core CODIS STR Loci 

D21S11D21S11 21q21.221q21.2 Ch.21; Ch.21; 19.476Mb19.476Mb

[TCTA][TCTG] [TCTA][TCTG] complexcomplex

1212--41.241.2 8282 3,4033,403

Typical DNA Profile

D3S1358D3S1358 14,1614,16 D13S317D13S317 11,1211,12

vWAvWA 14,1914,19 D7S820D7S820 10,1010,10

FGAFGA 21, 2121, 21 D16S539D16S539 12,1312,13

D8S1179D8S1179 11,1511,15 THO1THO1 7, 97, 9

D21S11D21S11 30, 3230, 32 TPOXTPOX 8,128,12

D18S51D18S51 15,1715,17 CSF1POCSF1PO 11,1311,13

D5S818D5S818 12,1212,12 AmelogininAmeloginin X,YX,Y

Multiplex PCR

• Over 15 Markers Can Be Copied at Once

• Sensitivities to levels less than 1 ng of DNA

• Ability to Handle Mixtures andAbility to Handle Mixtures and Degraded Samples

• Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

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310 Genetic AnalyzerCapillary Electrophoresis

Capillary Electrophoresis

Argon Ion LaserFill with Polymer Solution

50-100 μm x 27 cm

- +Capillary window

Inlet (cathode)

Outlet (anode)5-20 kV

Data Acquisition and Analysis

DNA Separation occurs in minutes...

CODIS Acceptable Kits

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AmpFlSTR® Profiler Plus IDTM System 

Current CODIS Acceptable Kits

AmpFlSTRAmpFlSTR®® COfilerCOfilerTMTM SystemSystem

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Probability of Identity

Profiler Plus® 1.48 x 10-11 1.04 x 10-11

African American US Caucasian

COfiler® 3.5 x 10-7 1.19 x 10-6

Profiler Plus / COfiler 1.8 x 10-15 3.8 x 10-14

CODIS DNA Profile

DCFBIWAD7

MD060215039Q4M

11,11;12,12;12,13;10,20;29,30;16,18;

12 13 09 11 11 15 23 24 06 9 3 08 0812,13;09,11;11,15;23,24;06,9.3;08,08;

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JCL

This is the only information sent to NDIS

1) ORI = DCFBIWAD7FBI Laboratory Wash. DC Div.7 

2) Specimen ID #  MD060215039Q4MMD, 2006, February 15 Case# 39 Question Specimen 4 Male Fraction

CODIS DNA Profile

Question Specimen 4 Male Fraction

3) STR Type11,11,12,12,12,13,10,20,29,30,16,18,12,13,09,11,11,15,23,24,06,9.3,08,08,17,19

4) Analyst Identifier = JCL

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References Available for nucDNA Analysis

Current CODIS Acceptable KitsCurrent CODIS Acceptable Kits

D8S1179 D21S11 D7S820 CSF1PO

D3S1358TH01

D13S317 D16S539 D2S1338

D19S433 D18S51TPOX

VWA

6FAM (blue)

VIC (green)

NED

AmpFlSTR® Identifiler™

D19S433 D18S51VWA

AMEL D5S818 FGA

GS500 LIZ size standard

NED (yellow)

PET (red)

LIZ (orange)

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Current CODIS Acceptable KitsCurrent CODIS Acceptable Kits

Reference Samples Available for Y‐Chromosome Analysis

Additional Multiplex STR Kits

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DYS456 DYS389 I DYS390 DYS389 II

DYS458 DYS19 DYS385 a/b

AmpFlSTR® Yfiler™ Kit 1 ng Male Control DNA 007

DYS393 DYS391 DYS439 DYS635 DYS392

Y GATA H4 DYS437 DYS438 DYS448

Challenges Associated With Nuclear DNA Analysis of Human Remains

Limited amounts of DNA availableLimited amounts of DNA availableDNA samples are often highly degradedDNA samples are often highly degradedPCR i hibit ft if ith DNAPCR i hibit ft if ith DNAPCR inhibitors often co purify with DNAPCR inhibitors often co purify with DNA

MiniSTR Development 

Smaller PCR products work better with low copy number or fragmented DNA templates

STR repeat regionminiSTRprimer

miniSTRprimer

Conventional PCR primer

Conventional PCR primer

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Additional Multiplex STR Kits

Mitochondrial DNA

Utility of mtDNA Analysis

BonesBonesTeethTeeth Cortical window: left tibiaCortical window: left tibiaTeethTeethHairsHairs

Cortical window: left tibiaCortical window: left tibia

TelogenTelogen Phase HairPhase Hair

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Advantages of mtDNA Analysis

• High copy number (several hundred to several thousand copies per cell) 

• Less prone to degradation due to structure and location

• Maternal inheritance (maternal relatives source of known sample in missing persons cases)

• Highly variable between individuals

References Available for mtDNA Analysis

mtDNA Architecture

D-loopp

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CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539D18S51

D8S1179

D3S1358

FGA VWA

AMEL

AMEL

CODIS + MitoCombines 13 Core STR Loci and mtDNA

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Sequence Analysis ofmtDNA Control Region

Sequence Electropherogram

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Sequence Data Analysis

• Data generated is analyzed using Sequencher™ Plus software.

• In order to be reported, there must be overlapping data from at least two amplifications or two extractions.

• Data is analyzed by two scientists independently to confirm results.

Sequence Comparison

Inheritance

• Where does our genetic material come from?• We get half of our genetic material from our mother, and half from our father

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References Available for nucDNA Analysis

PATERNITY TESTING

MOTHER

CHILD

ALLEGED FATHER

Two alleles for eachautosomal genetic marker

DNA Paternity Testing

• DNA testing is the most accurate and reliable means of identity verification available for parentage testing.

• A paternity or maternity test can prove with a 100% probability of exclusion that an individual IS NOT the biological parent of a child.

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Paternity Exclusion

X X X

Paternity Exclusion

X X X X

Paternity Exclusion

X X

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DNA Paternity Testing

• No test available can prove with a probability of paternity or maternity of 100% that a man or woman IS the biological parent of a child. 

• Currently available DNA testing will routinely id h 99 9999% b bili fprovide greater than a 99.9999% probability of 

paternity/maternity when the biological mother is tested in conjunction with the child and the alleged father. This is in excess of the 99% or 99.9% requirement of most U.S. civil courts. 

Paternity Inclusion

Paternity Inclusion

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Paternity Inclusion

AB

B

CD

C

Inheritance

BC

Children and parents will share alleles at each location in a DNA profile

AB

B

CD

C

AB

B

CD

D

Inheritance

Siblings may or may not share allelesat each location

AB

A

CD

C

AB

A

CD

D

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