Genetic Identity. Steve Harris SPASH - Biotechnology

Transcription

1 Genetic Identity Steve Harris SPASH - Biotechnology

2 Comparison of Organisms ORGANISM GENES BASE PAIRS Lambda Phage 40 50,000 E.coli 400 5,000,000 Yeast 13,000 15,000,000 Human 20,000 3,000,000,000 (3 billion)

3 Differences Human to human 0.1% Human to chimpanzee 2.0% Human to yeast 10.0%

4 Types of DNA Genomic-Linear, double stranded, very long (billions of base pairs (bp)) used to form chromosomes and genes (ordinary DNA-like your saliva sample) Plasmid-Circular, double stranded, short (3-7Kbp) (often used in genetic cloning) cdna-derived through the process of reverse transcription of RNA Mitochondrial-Found in the mitochondria of cells, only inherited from mother side (used for mapping populations and evolutionary separations)

5 Polymorphism Poly = Many Morph = Forms

6 Brief History of Forensic DNA Typing Ray White describes first polymorphic RFLP marker Alec Jeffreys discovers multilocus VNTR probes first paper on PCR FBI starts DNA casework first STR paper FSS starts UK DNA database FBI launches CODIS database

7 Techniques in Genetic Identity RFLP Restriction Fragment Length Polymorphism (restriction enzymes) SNP Single Nucleotide Polymorphism (one base change) STR Short Tandem Repeats (2-7 bases repeating) VNTR Variable Number of Tandem Repeats (7-200 bases repeating)

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

9 195 bp 170 bp TCAT repeat unit Different primer sets produce different PCR product sizes for the same STR allele

10 DNA Use in Forensic Cases Most are rape cases (>2 out of 3) Looking for match between evidence and suspect Must compare victim s DNA profile Challenges Mixtures must be resolved DNA is often degraded Inhibitors to PCR are often present

11 Human Identity Testing Forensic cases -- matching suspect with evidence Paternity testing -- identifying father Historical investigations Missing persons investigations Mass disasters -- putting pieces back together Military DNA dog tag Convicted felon DNA databases

12 Sources of Biological Evidence Blood Semen Saliva Urine & poo Hair Teeth Bone Tissue

13 Sample Obtained from Crime Scene or Paternity Investigation Steps in DNA Sample Processing Biology DNA Extraction DNA Quantitation PCR Amplification of Multiple STR markers Separation and Detection of PCR Products (STR Alleles) Technology Sample Genotype Determination Comparison of Sample Genotype to Other Sample Results Genetics Generation of Case Report with Probability of Random Match If match occurs, comparison of DNA profile to population databases

14 DNA in the Cell chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides

15 PCR Polymerase Chain Reaction Denature DNA (apply heat to DNA separates double stranded DNA into single stranded DNA. Anneal primers primers (synthetic DNA) attach above and below region of interest through complimentary base pairing. Extension of primers Taq Polymerase build new DNA by bringing in the correct complimentary base.

16 Scientists for a better PCR

17 DNA Amplification with the Polymerase Chain Reaction (PCR) Starting DNA Template Separate strands (denature) Forward primer Make copies (extend Add primers primers) 5 (anneal) 3 5 Reverse primer

18 PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

19 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 (we do not have Fluorescent dye capability)

20 ABI Prism 310 Genetic Analyzer capillary Syringe with polymer solution Injection electrode Outlet buffer Autosampler tray Inlet buffer

21 Close-up of ABI Prism 310 Sample Loading Area Electrode Capillary Sample Vials Autosampler Tray

22 Two different individuals Time-> Human Identity Testing with Multiplex STRs D3 amelogenin D8 TH01 D19 VWA D21 FGA AmpFlSTR SGM Plus kit DNA Size (base pairs) D16 D18 D2 probability of a random match: ~1 in 3 trillion amelogenin D3 D19 D8 VWA TH01 Results obtained in less than 5 hours with a spot of blood the size of a pinhead D21 FGA D16 D18 D2 Simultaneous Analysis of 10 STRs and Gender ID

23 STR genotyping is performed by comparison of sample data to allelic ladders Microvariant allele

24 Frequency STR Allele Frequencies Number of repeats TH01 Marker Caucasians (N=427) Blacks (N=414) Hispanics (N=414) *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

25 FBI s CODIS DNA Database Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1998 Links all 50 states Requires >4 RFLP markers and/or 13 core STR markers Current backlog of >600,000 samples

26 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 D5S818 FGA CSF1PO D8S1179 D7S820 TH01 VWA AMEL D13S317 D16S539 D18S51 D21S11 AMEL