1. DNA Testing Presented by : Zhang Xiufeng
Today I will give you a lecture about DNA testing
Presented by :
Zhang Xiufeng
From the department of Forensic Biology of
Kunming Medical University
Phone:

2. A general outline What is DNA and where is it found in our body?
What is Polymerase chain reaction (PCR) ?*
What is Polymerase chain reaction -short tandem repeat(PCR-STR) ?*
How to use the PCR-STR technique to solve the paternity test or individual identification ?
What is DNA sequence polymorphism and single nucleotide polymorphism(SNPs) ?
The following aspects are the contents of today’s course. Firstly, Firstly,we need to know , finally, we need to know

3. DNA DNA-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.

4. 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.

5. 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
He found the law of segregation and independent assortment

6. An individual’s DNA remains the same throughout life.
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.
And it will not change along with the environment
Owning to this characteristic,we can tell apart from each other.

7. Individual nucleotides
Now we look at the double DNA helix and the chromosome in the cell nucleus, this is the double stranded DNA molecule, it wound around the histone core surface to form nucleosome. The chromatin further condesed to form chromosome in order to fit into cells
Target Region for PCR
Individual nucleotides

8. Cell nuclear DNA
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.

9. Why DNA is so important? Two main tasks Individual identificition
If This boy want to know whether the bloodstain from the crime scene is identical to the dead women?
The child want to know whether this couple are his biological parents? Whose is his father or mother?Maybe somebody in your class will become a forensic biologist in future, how do you solve these problems?
After this class I hope every body could solve these problems use the knowledge we have learned today!
Individual identificition
Paternity test
Two main tasks

10. Now we look at another example, as we all know, in 2008, Wenchuan, a small city, was destroyed by the earthquake ,thousands of people lost their lives in this disaster, how can we putting the pieces back together? And how can you tell me or distinguish those dead people? From their appearance or their clothing? Sometimes maybe, but after a few days later, the corpse will be deeply decaied, at that time, whatcan we do ? Ihope everybody should study hard with these problems?
2008 Wenchuan Earthquake

11. There is only semen from both murder scenes.
1986. Two young girls, Lynda Mann and Dawn Ashworth, were sexually assaulted and then left brutally murdered in 1983 and 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.
If you encounter these problems, How can you find out the criminal?
Figure 12.12B

12. Maybe someone of your class will become a famous scientist oneday, how do you solve the problems.
Investigator at one of the crime scenes (above), Narborough, England (left)

13. 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

14. Now DNA testing have become the most accurate and powerful technology currently avaiable for determination of identity

15. Main steps of DNA testing
DNA extraction
Silver staining
PCR amplification
PAGE CE
fluorescence

16. How to identify substance that may contain DNA-Sources of Biological Evidence
Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Muscle
Fingerprint residus
Fingernail Clippings
Sloughed off cells

17. Sources of nonbiological Evidence
-Touching DNA
Cigarette Butts
Envelope & Stamps
Chewing Gum
Bite Marks
………
When you are smoking, the saliva will be left out the surface of cigarette butt
When we use saliva attach the stamp to the envelope, there will leave some sloughed off cells, DNA will there.

18. 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.

19. 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:
Broken the cell membrane, let DNA and other substance release into solution
Remove the binding protein and other redundant substances
Precipitation and purification of DNA
I will give you a detailed explaination in the laboratory class,

20. How to detect the target DNA you are interested in?
Interesting region
PCR technique

21. 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.

22. 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

23. The basic Principle of the PCR
Semi-conservative replication
The parental duplex is replicated to form two daughter duplexes, each of which consists of one parental strand, and one newly synthesized daughter strand, this behaviour called semi-conservative replication
many
few
The purpose of a (PCR) is to make a huge number of copies of a gene.

24. The basic Principle of the PCR
Denaturation 3’ 5’ 3’ 5’
By heating, when the temperature raise at 95, the hydrogen bands will be destroyed, so the double stranded DNA will be seperated into two single nucleotide.
95ºC 5’ 3’ 3’ 5’

25. The basic Principle of the PCR
Renaturation 5’ 3’
When the DNA temperature decreased at 20 degree, what happened to the DNA molecular? The two single strands DNA molecular can be bind eachother again, this process is called DNA renaturation. 3’ 5’
20ºC 3’ 5’ 3’ 5’

26. 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.
If you want to do a PCR ? What do you need? Target DNA as template, primers can bind to DNA templete,

27. What do you need-Requirements for PCR
3- Heat stable Taq DNA polymerase.
Enzyme that catalyzes the reaction
thermophillic 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.
The taq DNA polymerase can catalyzes the reaction, buffer solution contains magnesium.

28. How does it work-PCR Process
PCR –reaction is divided into 3 steps:
Which are repeated for 30 to 40 cycles
1-Denaturation: During denaturation,
the template DNA is seprated into its 2 separate by heating at the temperature about 95ºC.
Heat causes DNA
Heat causes DNA strands to separate, denaturation of DNA at 94
Destroy the hydrogen bands. 3’ 5’
95℃ 3’ 5’
95ºC 5’ 3’ 3’ 5’

29. 2-Anneling:
This involves the annealing of the primer to the denatured.
50-60 ℃
Primers anneal at oC 5’ 3’
When the temperature decreased to degree, what will happen? We know the primers in the mixtures of PCR reaction, the Primers will bind to the complementary sequence of target DNA.right? Ok, this process is called anneling. 3’ 5’ 5’ 3’ 3’ 5’

30. 3-Extension:
The synthesizing ,take place at a temperature of around 72ºC,this corresponds to the optimal temperature for the Tag-polymerase to work
50-60 ℃
Taq酶
Antisense
Primer
If we increased the temperature to 72 degree, what will happen? As we know the Taq DNA polymerase was obtained from the hotspring, the temperature was about 72 degree, 72 degree is a optimal temperature for this kind of process.the Taq DNA polymerase can catalyze the reaction by using dNTPS.so the single strand DNA can be extended, this process is called Extension
Sense
Primer
72℃
Taq酶

31. 72℃
The first cycle
At the end of the first cycle, what will happen? Compared with the previous one, we can get two double stranded DNA,moleculars. That means DNA duplicated doubles. Ok, if we repeat the first three steps, what will happen? We can get four DNA moleculars, right?

32. 72℃
The Second cycle
We can get four double stranded DNA moleculars at the end of the second cycle,ok

33. PCR的基本原理 20－30 cycles DNA template PCR反应条件 PCR过程 PCR的特点
So, if we repeated the denaturation, anneling, and extension for about cycles, what will happen? We will get thousands or millions of DNA moleculars, ringht?

34. PCR Technique
b1 = first new brown strand
g1= first new green strand

35. Millions copy of target DNA moleculars1 2 3 4 5 22 55 72 94
Time（min） T
（℃）
Denaturation 1
Annealing 2
Extension 3
Repeat steps
1～3
25～30 cycles
Millions copy of target DNA moleculars
DNA double helex

36. 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

37. Do you want to know what makes us different from each other?
Research discovered that 99% genomic DNA are identical among individuals, 1% of the genomic DNA is different among us, what makes us different from eachother. The answer is DNA polymorphism
The answer is DNA polymorphism

38. 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.

39. 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)

40. 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

41. 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.

42. Nomenclature for the gene locus and STR alleles
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

43. According to the Mendel‘s law, the law of segregation
and independent assortment, half part of all of the
child‘s genetic material from his father, the other
part must from his mother.
Schematic representation of two different STR loci on different pairs of homologous chromosomes. The chromosomes with the open circle centromeres are paternally inherited while the solid centromere chromosomes are maternally inherited. Thus, this individual
received the four repeat allele at locus A and the three repeat allele at locus B from their father, and the five repeat allele
at locus A and the six repeat allele at locus B from their mother

44. Short Tandem Repeats (STRs)
AATG
7 repeats 7 8
8 repeats
Heterozygote = alleles differ and can be resolved from one another
8 repeats
8 repeats 8
Homozygote = both alleles are the same length
the repeat region is variable among samples while the flanking regions where PCR primers bind are constant

45. Individual 1
Individual 2
Locus A
Locus B － ＋

46. The alleles are separated by the length of PCR products
Person 1
Person 2
Person 3
Person 4
Person 5
The alleles are separated by
the length of PCR products

47. 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)

48. Since humans are 99.9% identical where do crime scene investigators look for differences in DNA profiles?
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

49. Types of STR repeat sequences
Length of repeat unit
Number of repeats
The level of conformity of the repeated units

50. 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

51. Advantages of using tetranucleotide STR loci in forensic DNA typing
Conductive to multiplexing
Reduced allelic dropout
Capable of generating PCR product from degraded DNA samples
Reduced stutter product formation
Heterozygotes are easier to differentiate

52. The conformity of the repeated motif
Motif: the compound of the repeat unit
GATA
Simple Repeats---contain units identical in length and sequence
FES/FPS：[ATTT] PLA2A ：[ATT]8-17

53. The conformity of the repeated motif
Compound Repeats---contain two or more adjacent simple repeats
GABRB15：[GATA]5-12[GATC]2-4[TATC]1-2
TFIIDA：
[CAG]3[CAA]3[CAG]9-11CAA[CAGCAA]0-1[CAG]9-24[CAA]2

54. The conformity of the repeated motif
Complex Repeats---may contain several repeat blocks that vary in length or may have variable intervening sequences
D21S11：
TCTA/TCTG, bp
3 variable regoins and
1 constant regoin

55. 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

56. Nomenclature for STR alleles Choice of allele designation
According to the times of repeat unit
For complete repeat unit
Eg: TH01：[AATG]9
the times of repeat unit is 9，the allele named 9
[AATG] [AATG] [AATG] [AATG] [AATG] [AATG] [AATG] [AATG] [AATG] 1 2 3 4 5 6 7 8 9

57. Nomenclature for STR alleles Choice 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 nucleotides
The length of TH01 allele is 173bp：
[AATG]6ATG[AATG]3 ，the allele named 9.3

58. Desirable characteristics of STRs used in Forensic DNA typing
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. .

59. Desirable characteristics of STRs used in Forensic DNA typing
High variration among individuals
Low mutation rate
Separate chromosomal locations
Allele length range of bp
The number of allele is 8-12

60. 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

61. Application of DNA polymorphism-Analysis of Results: Who can’t be excluded?CS A B C D
AL: Allele ladder
CS: Crime Scene
A: Suspect A
B: Suspect B
C: Suspect C
D: Suspect D 15 10 7
Which suspect genotype matches that found at the crime scene?
Which suspect can’t be excluded?
BXP007 alleles 5 4 3 2 1
5-2
7-4
5-2
7-2
10-3
genotype

62. Genetic Inheritance Pattern of DNA Profiles
Father
Child
MOther

63. The application of PCR-STR in Paternity Testing
Family Inheritance of STR Alleles (D13S317)
PCR product size (bp) 11 14
Father
Results of DNA Tests Impact Families Me 12 14
Child #1 8 14
Child #2 11 12
Child #3 8 12
Mother

64. Paternity Testing Amanda Family Inheritance of STR Alleles (D13S317)
PCR product size (bp) 11 14
Father
Father
Marshall 12 14
Child #1 8 14
Child #2 11 12
Katy
Child #3 8 12
Mother
Mother

65. 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
It is usually 10 markers will be ,it will use different fluorescent dye to distinguish the STR locus,

66. 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.

67. 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.

68. PCR products have the same length , primers are labeled with different fluorescents
Different length of PCR products, primers are labeled with same flurescent

69. Information is tied together with multiplex PCR and data analysis
AmpFlSTR® Identifiler™ (Applied Biosystems)
D8S1179
D21S11
D7S820
CSF1PO
TH01
D13S317
D3S1358
D16S539
D2S1338
D19S433
VWA
TPOX
D18S51
D5S818
AMEL
FGA
1 integrated analysis vs. 16 separate runs

70. Capillary Electrophoresis Instrumentation
ABI capillary array
ABI single capillary

71. STR genotyping is performed by comparison of sample data to allelic ladders
What is allele ladders? A mixture of common alleles present in the human population for a particular marker,Generated with the same primers as the DNA sample and the reference DNA.it can be created within the lab or can be purchased by commmerical manufactures.
Profile of test subject
Reference standards with the known alleles for each STR locus

72. Fluorescent dye multiple locus composite amplificationAF C
How do we judge whether this child is this couples biological child? According to the mendelian pattern,one copy being inherited from his father, and the other copy must come from his mother, AM
One copy being inherited from his father, and the other copy coming from his mother

73. AF X Y 16 10 12 8 11 21 AM X X X X 16 18 10 16 8 19 21 C X 15 18 13 16 8 12 19

74. Crime Scene - Two Suspects
D3 vWA FGA
S1 14, , ,24
S2 15, , ,24
E 15, , ,24
Ok, let us look at this picture, the first line profile is the DNA sample from the suspect one, the second line profile is DNA sample from the suspect two, and the third line is the DNA sample from the crime scene, what can we see from this picture,at the gene locus D13S1358,at this locus we can see the suspect one have 14 and 15 alleles,how about the suscept two, he have 15 and 18 alleles, how about the crime scene profile at this locus, we can see this locus also contains 15 and 18 allele, ok ,now we look at the next locus,ok, so what can we get from this , the profile of the DNA sample from the crime scene consistent with the profile from suspect two.

75. AmpFlSTR® Identifiler ®

76. 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

77. DNA sequence polynorphism

78. SNPs(single nucleotide polymorphism)
Single nucleotide polymorphisms: regions of DNA where one base pair is different.
Occur evenly spread over all the DNA. 1/ 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 bp
In non-coding regoins every bp
Over 300,000 human SNPs known and are being mapped.
So it has the potential to be the next genetitation genetic mark

80. 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?

81. Reference STRBase Forensic DNA Typing， John Butler
NIST website:
STRBase