Jiang Qiang

Department of Forensic Medicine

Forensic DNA Typing  

Outline

1. Introduction

2. Application of forensic DNA typing

3. Process of forensic DNA typing

4. Conclusion

Brief History of Forensic DNA Typing

• 1980 - Ray White describes first polymorphic RFLP marker

• 1985 - Alec Jeffreys discovers multilocus VNTR probes

• 1985 - first paper on PCR• 1991 - first STR paper• 1995 - UK DNA database• 1998 – US CODIS database• 2000 – Other countries database

• DNA parting standardization

Examples of DNA in the News

2001 “911”, New York

2004Indian OceanTsunami

2008 Wenchuan Earthquake

What are we doing

IdentificationIdentification Paternity testingPaternity testing

How to do

• DNA analysis

• STR,Y-STR,X-STR

• mtDNA,SNP

Fingerprints have been used since 1901

Methods for Human Identification

DNA Fingerprints since 1985

Applications for Forensic DNA Typing

Forensic cases - matching suspect with evidence

Paternity testing - identifying father

Historical investigations

Missing persons investigations

Mass disasters - putting pieces back together

DNA database

Two main tasks of Forensic Biology

Individual Identification Paternity Testing

Polymerase Chain Reaction (PCR)

Locus

Allele

STR

Homozygote

Heterozygote

Basic Concepts

DNA in the Cell

chromosome

cell nucleus

Double stranded DNA molecule

Individual nucleotides

P C R( Polymerase Chain Reaction)

类似于 DNA的天然复制过程，在体外试管内，以一对特异性的寡核苷酸为引物，四种脱氧核苷酸为底物，待测 DNA为模板，在 DNA聚合酶的催化作用下，通过高温变性——低温退火——中温延伸的温度循环，选择性扩增特异性目的 DNA片段过程 。

1 2 3 4 5

22

55

72

94

时间（ min ）

温度

（℃）

适温延伸3

高温变性1

低温退火2

重复 1 ～ 3 步25 ～ 30 轮

目的 DNA 片段扩增 100 万倍以上

DNA 双螺旋

DNA 单链与引物复性

DN

A

变性

形成2

条单

链 子链延伸DNA 加倍

principle

Father’s Sperm

Mother’s Egg

Child’s Cell

For each inherited characteristic, an organism has two genes, one from each parent. We call these genes alleles.

基因座 (locus)

基因在染色体上的特定位置称基因座。

等位基因 (allele)

同一个基因座上的基因可以有多个，它们之间存在 DNA一级结构差异，导致产生相对性状，这种有差异的基因互称为等位基因。

A

B

个体 1 个体 2

－

＋

Short Tandem Repeats (STRs)

AATG

7 repeats

8 repeats

AATG

Homozygote: Alleles are identical on each chromosome ； both alleles are the same length

Heterozygote: Alleles differ on each on each chromosome ； alleles differ and can be resolved from one another

Fluorescent dye label

Fluorescent dye creates a labeled PCR product

Fluorescent dye creates a labeled PCR product

primer1

primer2primer1

primer2

number of repeat units

Locus is the region of the genome being examined

Sources of Biological EvidenceBlood/Blood stain

Semen/ Semen stain

Saliva/Saliva stain

Urine

Hair

Teeth

Bone

Tissue

Blood stainOnly a very small amount of sample is needed to obtain

a DNA profile

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

13 CODIS Core STR Loci

AMEL

AMEL

Sex-typing

Forensic STR Markers on Human Chromosomes

Sample Obtained from Crime Scene or

Paternity InvestigationBiology

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof Multiple STR

markers

PCR Amplificationof Multiple STR

markers

Technology

Separation and Detection of PCR Products(STR Alleles)

Sample Genotype

Determination

Genetics

Comparison of Sample Genotype to Other

Sample Results

Comparison of Sample Genotype to Other

Sample Results

If match occurs, comparison of DNA profile to population databases

If match occurs, comparison of DNA profile to population databases

Generation of Case Report with Probability

of Random Match

Generation of Case Report with Probability

of Random Match

Steps in Forensic DNA Typing

Make copies (extend primers)

Starting DNA Template

5’

5’

3’

3’

5’

5’

3’

3’

Add primers (anneal)

5’3’

3’5’

Forward primer

Reverse primer

Polymerase Chain Reaction (PCR)

Separate strands

(denature)

5’

5’3’

3’

PCR is a method of amplifying a specific region of the genome – go from 1 to over a billion copies in about several hours

Multiplex PCR• Over 10 Markers Can Be

Copied at Once• Sensitivities to levels less

than 1 ng of DNA• Ability to Handle Mixtures

and Degraded Samples• Different Fluorescent Dyes

Used to Distinguish STR Alleles with Overlapping Size Ranges

AMEL

D3S1358TH01

TPOX

D2S1338

D19S433

FGA

D21S11

D18S51

CSF1PO

D16S539

D7S820

D13S317

D5S818

VWA

D8S1179

1 integrated analysis vs. 16 separate runs1 integrated analysis vs. 16 separate runs

Information is tied together with multiplex PCR and data analysis

AmpFlSTR® Identifiler™ (Applied Biosystems)

ABI 3100 16-capillary array

ABI 310 single capillary

Capillary Electrophoresis Instrumentation

DAD

MOM

CHILD

Genetic Inheritance Pattern of DNA Profiles

Family Inheritance of STR Alleles (D13S317)

Father

Child #1

Child #2

Child #3

Mother

PCR product size (bp)

11 14

11

12 14

8 14

12

128

Me

PATERNITY TESTING

Results of DNA Tests Impact Families

Results of DNA Tests Impact Families

Family Inheritance of STR Alleles (D13S317)

Father

Child #1

Child #2

Child #3

Mother

PCR product size (bp)

11 14

11

12 14

8 14

12

128

Amanda

Marshall

Katy

Me

My Wife

PATERNITY TESTING

Crime Scene - Two Suspects

Suspect 1

Suspect 2

Evidence

D3 vWA FGA S1 14,15 17,18 23,24S2 15,18 17,19 23.2,24E 15,18 17,19 23.2,24

Main Tasks

Paternity TestingIndividual Identification

Conclusion

Forensic DNA Typing

The genome of each individual is unique (with the exception of identical twins)

Probe subsets of genetic variation in order to differentiate between individuals

DNA typing must be done efficiently and reproducibly (information must hold up in court)

Reference

• Forensic DNA Typing ， John Butler • NIST website: http://www.cstl.nist.gov/biotech/strbase

STRBase

Thank you for your attention!Thank you for your attention!谢谢各位同学谢谢各位同学 !!