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DNA STR Typing Sherri Phillips Washington State Patrol Crime Lab CODIS Unit email@example.com CODIS Combined DNA Index System • Used for linking serial crimes and unsolved cases with repeat offenders • Links DNA data in all 50 states • Searches are conducted routinely to see if there are matches between the DNA profile of a convicted offender and a crime scene sample or between crime scene samples. Levels of CODIS LDIS-Local DNA Index System • Tacoma, Marysville, Spokane, Vancouver and Seattle-casework SDIS-State DNA Index System • Located in Seattle at Crime Lab Headquarters NDIS-National DNA Index System • Administered by the FBI. Contains the qualifying DNA profiles from all fifty states. • CO sample must have all 13 loci, Forensic Unknowns must have 10 of 13 loci. CODIS Hits Hits in Washington State • 30 hits in 2008 2007 CODIS Hits by Crime Type (n=114) • 114 hits in 2007 • 99 hits in 2006 homicide 13% • 90 hits in 2005 other 8% burg/theft 33% • 139 hits in 2004 robbery 14% drugs 0% • 38 hits in 2003 assault 4% sex crimes 28% • 34 hits in 2002 • 22 hits in 2001 --As of 31 January 2008 • Tetra Nucleotide repeats • TPOX – [GAAT] • Allele designation of 12 mean the individual has 12 [GAAT] repeats Format for CO Samples • Buccal Swabs on FTA paper • Jails, Sheriffs Offices, DOC’s • Cotton swab → Foam swab • FTA paper- Cellulose based paper 4 chemical substances Cells lysed and immobilized Stable at room temp for several years Processing CO DNA samples Extraction Quantitation Quantifiler DNA IQ (Real-Time PCR) ABI 7000 Data Analysis Capillary Electrophoresis Amplification Identifiler Amp Kit GeneMapper 3130xl Genetic 9700 Thermal Software Analyzer Cycler Extraction (Manual) • Felon Hit Conformation, Out of Order samples and weak samples from Robot sets • DNA IQ kit- silica coated paramagnetic resin • DNA ↑ affinity for silica • Magnetic stand Quantitation • Real Time PCR (cycle to cycle change in fluorescents), amplifies hTERT locus • Taq man probe has a 5’ fluorescents reporter dye and a 3’ non fluorescents quencher • The probe anneals to sequence between forward and reverse primer. When Taq polymerase reaches the probe uses the 5’ endonuclease to digest the probe releasing the quencher from the fluorescents sequence • IPC non naturally occurring DNA sequence, detects presents of inhibitors • ABI PRISM 7000 Sequence Detection System instrument PCR • Identifier kit (5 dye) • FAM (Blue), VIC (Green), NED (yellow), PET (red), LIZ (Orange) • Kit amplifies 15 loci in one reaction • 30 primers! • 13 core CODIS loci plus Amelogenin, D2 and D19 • Hot Start PCR (Taq gold) chemically modified at a lysine residue, pH dependent • Below pH 7 modification falls off activating Taq • Tris Buffer- pH ↓ as temp ↑ • Initial 11 min incubation at 95°C Extraction (Robot) Universal Robot Extraction (Robot) cont. • QIAGEN BioRobot Universal Platform • Recently validated • 96 well plate 78 sample • Fully automated • Frees up time for data analysis • Human error rate and chance of contamination decreased • Pipetting is more accurate, faster • Normalization Capillary Electrophoresis Capillary Electrophoresis cont. • Automated procedure, minute amount of sample used • 15,000 volts • high surface area to volume ratio, ↑ heat dissipation compared to slab gel. • POP- Linear uncross-linked dimethyl polyacrylamide • Detection- time span of sample injection to sample detection by a laser. • Internal size standard and allelic ladder • Electrokinetic injection Genemapper ID • Software used to analyze CE results • Stutter (obts_011708) • pull up (mcs_013108, 1 Yellow) • tri allele (obts_012208) • microvarients (obts_012208) • dye blobs • spikes CODIS vs. Casework • Decide from a lot of evidence what is the most probative • Screening evidence, time consuming • Phenol chloroform (organic) extraction, time consuming • Differential extraction • Mixture interpretation • Statistic for testifying Casework Example • Angelita Axelson was found strangled and raped in a Seattle hotel room on June 18, 1981. • Semen was identified by the medical examiner. • In 2003, detectives in the SPD Cold Case unit submitted the evidence to the WSP Seattle Crime Lab for STR typing. Challenges of Decades Old Evidence • Vaginal swabs were submitted along with other evidence. • Brown swabs - usually means degraded DNA! • No DNA profile was obtained from these swabs. Is that all there is? (A very important question…..) • Further investigation revealed the presence of a vaginal smear prepared at the medical examiner’s office decades ago. Testing begins….. • The cover slip was “popped off” the vaginal smear and the residue was scraped into a tube. • The microscopic exam revealed the presence of spermatozoa. SPERM A STR/DNA Profile is Obtained • Upon completion of the DNA testing, nearly a full STR profile was obtained from the vaginal smear sperm fraction. • The DNA profile was searched in the convicted felon databank. A Hit to CODIS! • The DNA profile from the vaginal smear sperm fraction matches convicted felon Bryan Gelenaw- aka The Bus Stop Rapist. • When confronted with the DNA evidence, GUILTY! Bryan Gelenaw pled guilty to the murder of Angelita Axelson….
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