DNA TestingDNA TestingDNA TestingDNA TestingPresented byPresented by: :

Zhang XiufengZhang XiufengFrom the department of Forensic From the department of Forensic

Biology ofBiology of Kunming Medical UniversityKunming Medical University

Email:Email:xiuzheng1203@163.comxiuzheng1203@163.comPhone:15025149349Phone:15025149349

A general outline I. What is DNA and where is it found in our

body?

II. What is Polymerase chain reaction (PCR) ?*

III. What is Polymerase chain reaction -short tandem repeat(PCR-STR) ?*

IV. How to use the PCR-STR technique to solve the paternity test or individual identification ?

V. What is DNA sequence polymorphism and single nucleotide polymorphism(SNPs) ?

DNADNA-sometimes called”the genetic fingerprint”

Inherited from both parents, so biological connections can be confirmed.

1986-first used to convict an criminal of murder in England.

What is DNA? DNA is a double helix of two antiparallel

strands held together by hydrogen bonds

between complementary purine and pyrimidine

bases

The DNA strands are made up of four different

building blocks(A 、 T 、 G 、 C).

Double stranded DNA wound around the

histone core surface form repeating units

known as nucleosomes.

Genomic DNA further highly compacted in

order to to fit within cells.

What characteristics of DNA have?

DNA is the chemical substance which makes up our chromosomes and controls all inheritable traits (eye, hair and skin color)

DNA is different for every individual except identical twins

DNA is found in all cells with a nucleus (white blood cells, soft tissue cells, bone cells, hair root cells and sperm)

Half of a individual’s DNA/chromosomes come from the father & the other half from the mother-inherient in the mendelian pattern.

There are three billion base pairs in the genomic DNA

99% similarity among individuals, 1%

different DNA makes us different from

each other. In specific regions on a DNA

strand each person has a unique

sequence of DNA or genetic code.

An individual’s DNA remains the same

throughout life.

Target Region for PCRTarget Region for PCR

Individual nucleotides

The human genome contain 23 pairs of chromosomes in every cell.

Chromosomes 1-22 are called autosomes.

One copy being inherited from one‘s father, and the other copy coming from one‘s mother.

Sex-chromosomes are

either ( X ， Y ) for male, or ( X ， X ) for female.

Cell nuclear DNA

Paternity test

Individual identificition

Why DNA is so important?

Two main tasks

2008 Wenchuan Earthquake

1986. Two young girls, Lynda Mann and

Dawn Ashworth, were sexually assaulted

and then left brutally murdered in 1983 and

1986. Both murders occurred near the

village of Narborough in Leicestershire,

England with similar features leading the

police to suspect that the same man had

committed the crimes.

There is only semen from both murder

scenes. Figure 12.12B

Investigator at one of the crime scenes (above), Narborough, England (left)

Many useful Applications of Forensic Biology

Forensic cases-matching suspect with evidence

Paternity testing –identifying father or for those without birth certificates or the children out of wedlock or house hould registration.

Missing person investigations

Mass disasters—putting pieces back together

Historical investigations and genetic genealogy

Now DNA testing have become the

most accurate and powerful

technology currently avaiable for

determination of identity

Main steps of DNA testing

DNA extraction

PCR amplification PAGE

CE

Silver staining

fluorescence

• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Muscle• Fingerprint

residus• Fingernail

Clippings• Sloughed off

cells

How to identify substance that may contain DNA-Sources of Biological

Evidence

Sources of nonbiological Evidence-Touching DNA

Cigarette Butts

Envelope & Stamps

Chewing Gum

Bite Marks

………

Although these evidence are

nonbiological evidence, When a person

comes into contact with an object or

another person, a cross-transfer of

physical evidence can occur.

The intensity, duration, and nature of

the materials in contact determine the

extent of the transfer.

How to extract DNA from the biological evidence?

Organic DNA Extraction or Non-Organic DNA Extraction

Chelex DNA Extraction

Main steps of the extraction of DNA:

1.Broken the cell membrane, let DNA and other substance release into solution

2.Remove the binding protein and other redundant substances

3.Precipitation and purification of DNA

How to detect the target DNA you are interested in?

Interesting region

PCR technique

Definition of PCR

Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in vitro in approximately two hours.

PCR is just like DNA replication in

vivo.

It is the DNA synthesis reaction in

vitro, which using dNTP as

substrate, DNA as template, a pair

of oligonucleotides as primers, Taq

DNA polymerase as enzyme

The basic Principle of the PCR

Semi-conservative replication

The purpose of a (PCR) is to make a huge

number of copies of a gene.

fewmany

The basic Principle of the PCR

Denaturation

3’

5’

5’

3’

5’

3’

3’

5’

95ºC

The basic Principle of the PCR

Renaturation

5’

3’

3’

5’20ºC

3’

5’

5’

3’

What do you need-Requirements for PCR

1 -DNA sample :The DNA contains the sequence to be amplified Very small amount (ng or some times less).

2 -Two primers: oligonucleotides that define the sequence, 2 short specific pieces of DNA whose sequence flanks the target sequence,You must know the sequence of the flaking regions so you can order appropriate primers.

What do you need-Requirements for PCR

3 -Heat stable Taq DNA polymerase.Enzyme that catalyzes the reactionthermophillic enzyme from hot springs (Thermus aquaticus)

4-Four d NTPs. DNA building blocks, contains-dATP, dCTP, dGTP, dTTP

5 -Buffer(10x).maintains pH & contains salt, Magnesium chloride - enzyme cofactor

6-Thermocycler:Change temperature very rapidly for each cycle.

PCR –reaction is divided into 3 steps:

Which are repeated for 30 to 40 cycles1-Denaturation: During denaturation,

the template DNA is seprated into its 2 separate by heating at the temperature

about 95ºC.3’

5’

5’

3’

5’

3’

3’

5’

95ºC

95℃

How does it work-PCR Process

2-Anneling:

This involves the annealing of the primer to the denatured.

Primers anneal at 50- 60oC

5’

3’

3’

5’

3’ 5’3’5’ 50-60 ℃

50-60 ℃

72℃

Taq酶

Taq酶

Antisense

Primer

Sense

Primer

3-Extension:The synthesizing ,take place at a temperature of around 72ºC,this corresponds to the optimal temperature for the Tag-polymerase to work

72℃The first cycle

The Second cycle72℃

PCR的基本原理•PCR反应条件•PCR过程•PCR的特点

DNA template

20 － 30 cycles

PCR Technique

b1 = first new brown strand

g1= first new green strand

1 2 3 4 5

22

55

72

94

Time （ min）

T

（℃）

Extension

3

Denaturation

1

Annealing

2

Repeat steps 1 ～ 3

25 ～ 30 cycles

DNA double helex

Millions copy of target DNA moleculars

PCR has many applications

PCR is commonly used to produce many copies of a selected gene segment or locus of DNA.

In criminal forensics, PCR is used to amplify DNA evidence from small samples that may have been left at a crime scene.

PCR can be used to amplify DNA for genetic disease screening

Do you want to know what makes us different from each

other?

The answer is DNA polymorphismThe answer is DNA polymorphism

What is DNA polymorphism?

Definition: more than one normal allele at a gene locus in the population, with the rarest allele exceeding a frequency of 1%.

Characteristics:

The frequency of the rarest allele is more than

1%.

Inherited in Mendelian pattern in the families.

No functional consequences.

The major types of DNA polymorphism

Short tandem repeat polymorphism(STR)*

Restriction fragment length polymorphism (RFLP)

DNA length polymorphism

DNA sequence polymorphism

Single nucleotide polymorphism(SNP)

Short tandem repeat(STR)

Repeating units of an identical (or)similar

DNA sequence, where the repeat sequence is

2-6 base pairs in length. The repeat units are

arranged in direct succession of each other,

and the number of repeat units varies

between individuals.

Repeating unit Repeating unit Repeating unit

What is PCR-STR technique?Isolation of STR markers using PCR technique

Schematic of STR DNA markers. PCR primers are designed to target invariant flanking sequence regions. The number of tandem repeat units in the repeat regions varies among individuals making them useful markers for human identification.

According to the times of repeat unit

Locus or loci: refers to the location on the chromosome

Allele: refers to the type of DNA for STRs, the allele is the

number of repeats

Each person has two copies of each chromosome, so each

person has 2 alleles at each locus

Nomenclature for the gene locus and STR alleles

According to the Mendel‘s law, the law of segregationand independent assortment, half part of all of thechild‘s genetic material from his father, the other part must from his mother.

Short Tandem Repeats (STRs)

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

7 repeats

8 repeats

AATG

Homozygote = both alleles are the same length

7 8

Heterozygote = alleles differ and can be resolved from one another

8 repeats

8 repeats 8

Locus A

Locus B

Individual 1 Individual 2

－

＋

Person 1

Person 2

Person 3

Person 4

The alleles are separated by

the length of PCR products

Person 5

Methods for identification of STR markers

Search DNA sequence databases such as Genbank with more than six or so contiguous repeat units(Weber & May, 1989)

Perform molecular biology isolation methods(Edwards et al, 1991)

Crime Scene Investigators search in areas

of the genome that are unique from

individual to individual and are

“anonymous” (control no known trait or

function) The areas examined are Short

Tandem Repeats or STR’s

STR regoins

Since

humans are

99.9%

identical

where do

crime scene

investigators

look for

differences in

DNA profiles?

Types of STR repeat sequences

Length of repeat unit

Number of repeats

The level of conformity of the

repeated units

length of repeat unit

Mononucleotide repeats

Dinucleotide repeats

Trinucleotide repeats

Tetranucleotide repeats

Tetranucleotide repeats---have become the most popular markers for human identification(AGAT or GATA) in forensic biology

Advantages of using tetranucleotide STR loci in

forensic DNA typing

Conductive to multiplexingReduced allelic dropoutCapable of generating PCR product

from degraded DNA samplesReduced stutter product formationHeterozygotes are easier to

differentiate

The conformity of the repeated motif

Simple Repeats---contain units identical in length and

sequence

FES/FPSFES/FPS ：： [ATTT][ATTT]8-14 8-14 PLA2A PLA2A ：： [ATT][ATT]8-178-17

Motif: the compound of the repeat unit

GATA

Compound Repeats---contain two or more

adjacent simple repeats

GABRB15GABRB15 ：： [GATA][GATA]5-125-12[GATC][GATC]2-42-4[TATC][TATC]1-21-2

TFIIDATFIIDA：：[CAG][CAG]33[CAA][CAA]33[CAG][CAG]9-119-11CAA[CAGCAA]CAA[CAGCAA]0-0-

11[CAG][CAG]9-249-24[CAA][CAA]22

The conformity of the repeated motif

Complex Repeats---may contain several repeat blocks

that vary in length or may have variable intervening

sequences

D21S11D21S11 ：：

TCTA/TCTG, 172-264bpTCTA/TCTG, 172-264bp 33 variable regoins andvariable regoins and 1 constant regoin1 constant regoin

The conformity of the repeated motif

Nomenclature for STR loci

Intron STRs –use coding strand

Intergenic DNA-use strand first described in literature of database entry(Genbank)

First motif that can define the repeat is used

According to the times of repeat According to the times of repeat unitunit

For complete repeat unitFor complete repeat unit

EgEg: TH01TH01 ：： [AATG][AATG]99

the times of repeat unit is the times of repeat unit is 99 ，， the allele the allele

named named 99

[AATG] [AATG] [AATG] [AATG] [AATG] [AATG] [AATG] [AATG]

[AATG]

Nomenclature for STR allelesChoice of allele designation

1 2 3 4 5 6 7 8 9

Nomenclature for STR allelesChoice of allele designation

Some allele will have microvariant:

A common microvariant of TH01 （ Number of repeats ） ·（ Number of bp in incomplete repeat ）9 tetranucleotide repeats and one incomplete repeat of three nucleotidesThe length of TH01 allele is 173bp ：

[AATG]6ATG[AATG]3 ， the allele named 9.3

STR markers are easily to amplified. Smaller size advantageous where degraded DNA is common.

Thousands of polymorphic STR loci have been identified in human genome.

STR markers are scattered throughout the genome.

.

Desirable characteristics of STRs used in Forensic DNA typing

Desirable characteristics of STRs used in Forensic DNA typing

High variration among individuals

Low mutation rate

Separate chromosomal locations

Allele length range of 90-500bp

The number of allele is 8-12

13 have been chosen for use in forensic work

The 13 independently assort, meaning they are

on different chromosomes or far apart on the

same. Product law can be used

Each of the 13 have a number of different

alleles, Alleles differ by number of repeats

107

5

4

3

2

1

CS A B C D

genotype

5-2 7-4 5-2 7-2 10-3

AL: Allele ladderCS: Crime SceneA: Suspect AB: Suspect BC: Suspect CD: Suspect D

AL

15

BX

P007 a

llele

s

Application of DNA polymorphism-Analysis of Results:

Who can’t be excluded?

Father

MOther

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

The application of PCR-STR in 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

Father

Mother

Paternity Testing

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

Multiplex PCR

STRs are isolated using PCR

Primers have been development to allow amplification of multiple STR loci in a single reaction mixture.

Each primer set has been optimized such that its product, no matter the number of STRs, is not the same size as any of the other products.

Each primer set has unique fluorescent molecules covalently linked to them so that they may be visualized immediately by a computer.

STRs are isolated using PCR

Following the PCR reaction, internal DNA length standards are added to the PCR reaction mixture.

The DNAs are separated by length in a capillary gel electrophoresis machine

As DNA peaks elute from the gel they are detected with laser activation.

The results are then graphed by a computer which compares them to a standard.

PCR products have the same length ,

primers are labeled with different

fluorescents

Different length of PCR products, primers

are labeled with same flurescent

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

STR genotyping is performed by comparison of sample data to allelic

ladders

Reference standards with the known alleles for each STR locus

Profile of test subject

One copy being inherited from his father, and the other copy coming from his mother

Fluorescent dye multiple locus composite amplification

AF

C

AM

AF

AM

C

XY 16 1012 8 11 21

16 18 10 16 8 19 21X

X 15 18 13 16 8 12 19

XX X

Crime Scene - Two Suspects

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

AmpFlSTR® Identifiler ®

The advantages about the application of STR DNA typing in

Forensic biology

High sensitivity-very small

amount(ng or less)

High discriminating ability-(>99.99%)

Standardization, automatic typing

DNA sequence polynorphism

Single nucleotide polymorphisms: regions of DNA where one base pair is different.

Occur evenly spread over all the DNA. 1/ 1000-3000 bp-the most simple form and the most common source of genetic polymorphism in the human genome(90%)

SNPs occurs in human could be in coding or non-coding regoins:In coding regoins every 1000-3000bpIn non-coding regoins every 500-1000bp

Over 300,000 human SNPs known and are being mapped.

SNPs(single nucleotide polymorphism)

1 please find out at least 5 kinds biological evidence that contains DNA.

2 what do you need to do a PCR?

3 what is the principle and the process of PCR?

4 What is short tandem repets(STRs)?

5 what is heterozygote and homozygote?

6 How can you use the PCR-STR technique to solve the paternity test and individual identification problems?

Reference

STRBase

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