Forensic Biology by Richard Li

1. Chapter 20.1-20.2.1: DQA1/PM Chapter 18: Autosomal STR Profiling Forensic Biologyby Richard Li

2. DQA1-PM PCR-based Typing • Based on SNPs (single nucleotide polymorphisms) • Variations in the human genome • Single-base pair change originating from spontaneous mutations • Majority of human DNA polymorphisms • 1.4 million identified • Most are bi-allelic (e.g. AGT and ATT are common alleles but ACT and AAT are not)

3. Basic Characteristics of SNPs • Advantages: • SNPs are abundant and can be used as markers • Can easily be amplified by PCR • 50-100 bp in length • No “binning” or statistical problems • Useful for phenotyping • Disadvantages: • Not very polymorphic (most are only dimorphic) • Much lower level of discrimination than DNA Fingerprinting or STR analysis

4. Forensic Applications • Very important during late 1980’s and 1990’s in forensic labs (before discovery of STRs) • PCR-based: • Allowed for analysis of trace evidence and degraded samples • Loci: • HLA-DQA1 (Average Pm = 1/20) • Polymarker (Average Pm = 1/164) • Combined average Pm = 1/3,300

5. Forensic Applications • Methods to detect SNPs: • DNA sequencing (labor-intensive) • Allele-specific oligonucleotide (ASO) hybridization assays (fast and easy) • ASO probes hybridize to their complementary DNA sequences to distinguish known polymorphic alleles • Steps: • DNA extraction • PCR amplification across the SNP site using biotinylated primers • Denaturation of DNA hybridization to immobilized probes • Colorimetric detection

11. STR Typing: Basics • Like VNTRs, are tandem repeats • Length of repeat motif is less than 10 bp • Also known as “microsatellites” • Block is usually less than 500 bp • Advantages: • Lots of them (more than 100,000 in human genome) • Very polymorphic • Block is small enough for PCR amplification • Good for trace evidence and degraded DNA • Amplicons can be separated on high resolution polyacrylamide gels

14. Characteristics of STR Loci • Repeat Unit Length • Dimeric (CT), Trimeric (CAT), Tetrameric (CTTG) ... • High degree of polymorphism • Mutation “hot spots” • Located in intergenic DNA • Micorvariants (e.g. 15.2) • Population Match Probability • Best STR systems: • Highly polymorphic STRs • Relatively equal frequency distribution at all loci • Unlinked

15. STR Commonly Used • STR multiplex system in U.K.- 4 loci • 1995- first national database established in U.K. • 6 STR loci + amelogenin (4 loci added later) • 1997- first database in U.S. • CODIS • FBI • 13 core loci + amelogenin • 2 more now added for a total of 15

16. Forensic STR Analysis • Loci are amplified using fluorescent dye-labeled primers • Separated using polyacrylamide electrophoresis • Detection: • Wavelength of fluorescence • Time to window • Amplitude of signal • Results in an electropherogram • Size of each amplicon determined by comparison to internal size standard (ROX, LIZ)

17. Relative fluorescent units (rfu’s) Time since injection = amplicon length

18. Factors Affecting Genotyping Results • Mutations at STR core repeat regions • During embryogenesis • During spermatogenesis • Duplications • Primer binding site mutations • Amplification artifacts • Allelic drop out , allelic drop in, stutter • Electrophoretic artifacts • Pull-up, dye blobs, and spikes

19. Genotyping of Challenging Forensic Samples • Degraded DNA • MiniSTR multiplex kits • Low-copy Number DNA (LCN) • < 100 pg of DNA • Mixtures • Sexual assault cases • Mixture interpretation

20. Interpretation of Results • SWGDAM & DNA Commission of the ISFG: • Inclusion (Match) • Calculate Pm • Sometimes challenged in Court (especially mixtures) • Exclusion • No calculation needed • Sometimes challenged in Court (especially mixtures) • Inconclusive • Multiple interpretations may be possible • Often challenged in Court