2.  Types of STR markers- 5 types based on sequence  STR allele nomenclature  Allelic ladder  Serological methods of identity profiling  Identity profiling based on DNA

3.  Two methods: RFLP & PCR based  Sir Jeffrey’s multi-locus RFLP on VNTR  Weeks to complete but still better than previous methods  RFLP: Restriction Fragment Length Polymorphism  Based on bacterial restriction enzymes  The most commonly used restriction enzyme in the United States was Hae III

4.  A sequence of DNA having a restriction site on each end with a "target" sequence in between  A target sequence is any segment of DNA that bind to a probe by forming complementary base pairs  A probe is a sequence of single-stranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be detected  When a probe base pairs to its target, it can be detected by its tag  RFLP produces a series of bands when a Southern blot is performed with a particular combination of restriction enzyme and probe sequence

6.  DNA extraction  Restriction enzyme digestion  Gel electrophoresis  Southern blotting  Probe binding and visualization (may include several steps for multiple loci)  Genotype determination

8.  In the original Jeffreys method, a single probe labeled multiple VNTR loci  Single-locus probes- one (homozygote)/two (heterozygote) alleles were detected

10.  Measures variation at the level of DNA sequence, not protein sequence  Requires very little background knowledge of DNA sequence BUT  Large initial test sample (>50 ng)  Many copies of any fragment to get detectable signal  No replication (“amplification”) of original sample in this technique  If sample is degraded, and some strands lost, signal strength problem (above) worsens  If degradation includes breaking of strands (likely) => New fragments NOT DUE TO RESTRICTION SITES= Adding new FALSE lines to fingerprint!!

11.  Problem with RFLP  Problem with VNTR  Problem with acceptability  Check O. J. Simpson murder case  PCR came in

12.  Polymerase chain reaction. By Kary Mullis in 1983  Based on DNA replication process of cells  Involves bracketing a certain sequence on both sides with primers and using enzymes to copy (amplify) that sequence multiple times

14.  HLA DQ alpha/DQA1, chromosome 6, SSO probes bound at specific locations on a test strip composed of nylon membrane  PolyMarker (PM+DQA1) coamplified a portion of the HLA DQ alpha gene along with five other DNA segments located on human chromosomes 4, 7, 11, and 19  D1S80, a PCR-amplified VNTR on chromosome 1 containing a 16-bp repeat unit and alleles spanning the range of 14 to 41 repeat units. The PCR products from D1S80 ranged from approximately 400 to 800 bp and were typically separated on a vertical polyacrylamide gel followed by silver-stain detection  Short tandem repeats (STRs) discovered in the late 1980s, at about the same time as D1S80 and other minisatellite

16.  When all 13 CODIS core loci are tested, the average random match probability is rarer than one in a trillion among unrelated individuals  the 13 CODIS core STR loci may be divided up into four categories: 1. Simple repeats consisting of one repeating sequence: TPOX, CSF1PO, D5S818, D13S317, D16S539; 2. Simple repeats with non-consensus alleles (e.g., 9.3): TH01, D18S51, D7S820; 3. Compound repeats with non-consensus alleles: VWA, FGA, D3S1358, D8S1179; 4. Complex repeats: D21S11.

19.  DNA Extraction  DNA Quantitation  PCR amplification of multiple STR loci (10 – 15)  Separation of PCR product by capillary electrophoresis  Data collection  Peak identification  Colour separation  Peak sizing  Comparison with allelic ladder  Genotype assignment  DNA profile

20.  Organic Extraction  Chelex extraction  FTA Card  Solid phase extraction- Qiagen columns, DNA IQ, PrepFiler  Differential extraction

22.  Fundamentals of Forensic DNA Typing- John M. Butler  Forensic DNA Typing, Biology, Technology, and Genetics of STR Markers- John M. Butler- 2nd edition