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:[email protected]@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