STR Interpretation Review

STR Interpretation Review - MCB 3416, Fall 2008 September...

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Unformatted text preview: MCB 3416, Fall 2008 September 18, 2008 y y y y E y y 7DT SRO\PHUDVH ZLOO RIWHQ DGG DQ H[WUD QXFOHRWLGH y y WR WKH HQG RI D 3&5 SURGXFW PRVW RIWHQ DQ $ y 'HSHQGHQW RQ HQG RI WKH SULPHU y &DQ EH HQKDQFHG ZLWK H[WHQVLRQ VRDN DW WKH HQG RI WKH 3&5 F\FOH H J PLQ # RU R& y &DQ EH UHGXFHG ZLWK QHZ SRO\PHUDVH y %HVW LI WKHUH LV 127 D PL[WXUH RI $ SHDNV A A Non-Template Addition (+A) vs. Minus A (-A) When you can't beat nature... help it. Taq polymerase tends to add a single non template A to the end of all amplification products. But some failures. 9 Another "undesired PCR product" 9 Reproducible feature of Taq activity. RFU -A True Allele Examples of A Peaks 100 101 Size (bp) 9 PCR systems are optimized for maximum +A. 9All Ladder peaks are +A Stutter Stutter a reproducible characteristic of Taq activity. Case Examples of Stutter Alleles called Stutter is the result of repeat slippage during the primer extension step of PCR. Mechanism: Taq polymerase can stall and fall off during DNA synthesis. The extended stand breathes apart and can reanneal out of register. The most common result is a minor peak at N-4 base pairs. RFU Below Stutter Threshold 100 104 Size (bp) Spectral Overlap Leads to Pull-Up Examples of Pull-up The light emitted from one fluorescent dye overlaps into other (wavelengths) colors. This overlap needs to be corrected.. Gray boxes represent the wavelength range used to measure fluorescence from each dye. > K y y ^ Item # 1CA 1CB 4SA 4SB 2 3-S1 15,18 15,17 16,19 16,17 23,25 22 X X,Y 14 13,15 28,33.2 29 16,17 12,16 12,13 11,13 9,13 11 12 8,9 D3S1358 vWA FGA AMEL D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 y y y y y y Item # 1CA 1CB 4SA 4SB 2 3-S1 D3S1358 D16S539 AMEL THO1 TPOX CSF1PO D7S820 15,18 15,17 11 10,13 X X,Y 6,9 9,9.3 10,12 9,11 10,11 11,12 12 8,9 AmpFlSTR IdentifilerTM (Applied Biosystems) d d < D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 y D19S433 VWA TPOX D18S51 y AMEL D5S818 FGA 1 integrated analysis 1 integrated analysis vs. 16 separate runs vs. 16 separate runs D y 1RW DOO DOOHOHV KDYH IXOO OHQJWK UHSHDW XQLWV y $OOHOHV ZLWK SDUWLDO UHSHDW XQLWV DUH GHVLJQDWHG E\ WKH QXPEHU RI IXOO UHSHDWV DQG WKHQ D GHFLPDO SRLQW IROORZHG E\ WKH QXPEHU RI EDVHV LQ WKH SDUWLDO UHSHDW y ([DPSOH 7+ DOOHOH 1 2 3 4 5 ATTTCCCT AATG AATG AATG AATG AATG TH01 9.3 allele AATG ATG AATG AATG AATG CACCATGGAGT 6 .3 7 8 9 D y 'HILQHG DV DOOHOHV WKDW DUH QRW H[DFW PXOWLSOHV RI d y y y y WKH EDVLF UHSHDW PRWLI RU VHTXHQFH YDULDQWV RI WKH UHSHDW PRWLI RU ERWK 6DLG WR EH FDXVHG E\ VOLSSDJH GXULQJ UHSOLFDWLRQ 0D\ H[LVW DV LQVHUWLRQ GHOHWLRQ RU EDVH FKDQJH 6HTXHQFH YDULDWLRQ FDQ RFFXU ZLWKLQ UHSHDW LQ WKH IODQNLQJ UHJLRQ RU LQ D SULPHU ELQGLQJ VLWH &DQ FDXVH 3&5 IDLOXUH GXH WR SRO\PRUSKLVP LQ WKH SULPHU VLWH QXOO DOOHOHV y y Off Ladder Alleles ^ W W D y y Z&h Shoulder Peak: always on the right y y t d / D / / / D Z&h / Z&h D ^ D Z&h W ^ K / / y y y y y y y Mixture interpretation The factors that can effect outcome: 1. Absolute quantity of DNA 2. Ratio of major/minor contributor 3. Number of contributors 4. Allele sharing among contributors 5. Extent of degradation 6. Three allele patterns 7. Taq activity is semi-quantitative 8. Primer site microvariants 1 + 5--impact stochastic effects Profile A Detected Factors: 1. Quantity 2. Quality Profile B Detected / 25:1 1:1 y Only Profile B Detected Profiles A and B Detected Factors: 1. Quantity 2. Quality Ratio DNA Mixture from a Burglary DNA Mixture from a Sexual Assault / y / y y DNA Mixture from a Burglary AMEL D19 D3 D8 TH01 VWA 15 years old (room temp storage) "Decay curve" of degraded DNA D21 FGA D16 D18 D2 D3 AMEL D19 D8 VWA D21 TH01 6 years old (-20 oC storage) D16 FGA D18 D2 Results with SGM Plus STR kit (Applied Biosystems) / y Item # 1S vic 1CA 1CB D3S1358 17,18 17,18 15,16,17,18 vWA 14,18 14,18 14,18,19 FGA 19,23 19,23 20,23 AMEL X X X,Y D8S1179 14 14 13,14 D21S11 30,31.2 30,31.2 28,29,30 31.2 28,29 D18S51 12,16 12,16 12,16,18, 20 18,20 D5S818 11,12 11,12 11,12, 13 12,13 D13S317 10,11 10,11 9,10,11, 12 9,12 D7S820 7,12 7,12 10,12 (7*) y y 2 sus 15,16 18,19 20 X,Y 13,14 10,12 Item # 1S vic 1CA D3S1358 17,18 17,18 15,16,17,18 15,16 D16S539 12 12 10,12 10,12 AMEL X X X,Y X,Y THO1 7,9 7,9 7,8,9 8,9 TPOX 8,10 8,10 8,10,12 8,12 CSF1PO 10 10 10 10 D7S820 7,12 7,12 10,12, (7*) 10,12 y 1CB 2 sus * = additional minor peak(s) detected <50 RFU but >30 RFU 1CA = vaginal swab, epithelial-rich fraction 1CB = vaginal swab, sperm-rich fraction Item # 1S vic 1CA 1CB D3S1358 17,18 17,18 15,16,17,18 vWA 14,18 14,18 14,18,19 FGA 19,23 19,23 20,23 AMEL X X X,Y D8S1179 14 14 13,14 D21S11 30,31.2 30,31.2 28,29,30 31.2 28,29 D18S51 12,16 12,16 12,16,18, 20 18,20 D5S818 11,12 11,12 11,12, 13 12,13 D13S317 10,11 10,11 9,10,11, 12 9,12 D7S820 7,12 7,12 10,12 (7*) Item # 1S vic 1CA 1CB D3S1358 17,18 17,18 15,16,17,18 vWA 14,18 14,18 14,18,19 FGA 19,23 19,23 20,23 AMEL X X X,Y D8S1179 14 14 13,14 D21S11 30,31.2 30,31.2 28,29,30 31.2 28,29 D18S51 12,16 12,16 12,16,18, 20 18,20 D5S818 11,12 11,12 11,12, 13 12,13 D13S317 10,11 10,11 9,10,11, 12 9,12 D7S820 7,12 7,12 10,12 (7*) 2 sus 15,16 18,19 20 X,Y 13,14 10,12 2 sus 15,16 18,19 20 X,Y 13,14 10,12 Item # 1S vic 1CA 1CB 2 sus D3S1358 17,18 17,18 15,16,17,18 15,16 D16S539 12 12 10,12 10,12 AMEL X X X,Y X,Y THO1 7,9 7,9 7,8,9 8,9 TPOX 8,10 8,10 8,10,12 8,12 CSF1PO 10 10 10 10 D7S820 7,12 7,12 10,12, (7*) 10,12 Item # 1S vic 1CA 1CB 2 sus D3S1358 17,18 17,18 15,16,17,18 15,16 D16S539 12 12 10,12 10,12 AMEL X X X,Y X,Y THO1 7,9 7,9 7,8,9 8,9 TPOX 8,10 8,10 8,10,12 8,12 CSF1PO 10 10 10 10 D7S820 7,12 7,12 10,12, (7*) 10,12 SAMPLE INTERPRETATION 1. Item # 1CA = single source 1S vic (Victim) is included as being the source of the sample profile at all loci 2 sus (Suspect) is excluded as being the source of the sample profile 2. Item # 1CB = mixture of 2 or more individuals 1S vic cannot be eliminated as a contributor to the sample profile 2 sus is included as being a contributor to the sample profile at all loci 1. Item # 1CA = single source Random Match Probability include all loci STATISTICS 2. Item # 1CB = mixture of 2 or more individuals Combined Probability of Inclusion exclude locus FGA and D7S820 7KH ([DPLQDWLRQ RI %LRORJLFDO (YLGHQFH 6FUHHQLQJ PHWKRGV DQG &RQILUPDWLRQ RI ERG\ IOXLGV 7\SHV RI %RG\ )OXLGV (QFRXQWHUHG LQ &ULPLQDO &DVHV ;6HPHQ 6HPLQDO )OXLG ;6DOLYD ;8ULQH ;9DJLQDO 6HFUHWLRQV ;)HFDO 0DWHULDO ;3HUVSLUDWLRQ ;+XPDQ 0LON 7\SHV RI &ULPHV ,QYROYLQJ %RG\ )OXLGV )6H[XDO $VVDXOWV )$VVDXOWV )+RPLFLGHV )%XUJODULHV )7KUHDWHQLQJ +DUDVVPHQW )'HVHFUDWLRQV :K\ 6KRXOG %RG\ )OXLGV %H ,GHQWLILHG" 7R KHOS SURYH DQ HOHPHQW RI D FULPH 7R SURYLGH D OLQNDJH EHWZHHQ LQGLYLGXDOV WKURXJK WKH LGHQWLILFDWLRQ RI JHQHWLF PDUNHUV 7R KHOS LQWHUSUHW JHQHWLF GDWD PL[WXUHV 7R DVVLVW LQ WKH UHFRQVWUXFWLRQ RI HYHQWV Identification of Blood 1. Chemical screening test (Kastle-Meyer). + result = could be blood. phenolphthalin + Hb reduced (colorless) H2O2 6HPHQ 6HPLQDO )OXLG 91RUPDOO\ FRQWDLQV VSHUPDWR]RD DQG WKH VHFUHWLRQV RI WKH VHPLQDO YHVLFOHV SURVWDWH JODQG DQG EXOERXUHWKUDO JODQG 91RUPDO HMDFXODWH YROXPH 9 PO phenolphthalein oxidized (pink) 2. Confirmatory test. Detect human hemoglobin by Ouchterlony or rapid immunoassay. 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