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 Email:xiuzheng1203@163.com Phone:15025149349

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

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.

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 He found the law of segregation and independent assortment

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.

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

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.

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

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

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 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. If you encounter these problems, How can you find out the criminal? Figure 12.12B

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)

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 Silver staining PCR amplification PAGE CE fluorescence

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

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.

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: 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,

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 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.

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’

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’

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,

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.

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’

2-Anneling: This involves the annealing of the primer to the denatured. 50-60 ℃ Primers anneal at 50- 60oC 5’ 3’ When the temperature decreased to 50-60 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’

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

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?

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

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 20-30 cycles, what will happen? We will get thousands or millions of DNA moleculars, ringht?

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

Millions copy of target DNA moleculars 1 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

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? 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

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.

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

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

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

Individual 1 Individual 2 Locus A Locus B － ＋

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

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)

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

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 multiplexing Reduced allelic dropout Capable of generating PCR product from degraded DNA samples Reduced stutter product formation Heterozygotes are easier to differentiate

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]8-14 PLA2A ：[ATT]8-17

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

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, 172-264bp 3 variable regoins and 1 constant regoin

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

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

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

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. .

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

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

Genetic Inheritance Pattern of DNA Profiles Father Child MOther

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

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

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,

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

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

Capillary Electrophoresis Instrumentation ABI 3100 16-capillary array ABI 310 single capillary

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

Fluorescent dye multiple locus composite amplification AF 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

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

Crime Scene - Two Suspects D3 vWA FGA S1 14,15 17,18 23,24 S2 15,18 17,19 23.2,24 E 15,18 17,19 23.2,24 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.

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

SNPs(single nucleotide polymorphism) 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 1000-3000bp In non-coding regoins every 500-1000bp Over 300,000 human SNPs known and are being mapped. So it has the potential to be the next genetitation genetic mark

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 STRBase