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Contribution of the National Institute of Standards and Technology, 2010 Final Figures Fundamentals of Forensic DNA Typing Forensic DNA Typing, 3 rd Edition:

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Presentation on theme: "Contribution of the National Institute of Standards and Technology, 2010 Final Figures Fundamentals of Forensic DNA Typing Forensic DNA Typing, 3 rd Edition:"— Presentation transcript:

1 Contribution of the National Institute of Standards and Technology, 2010 Final Figures Fundamentals of Forensic DNA Typing Forensic DNA Typing, 3 rd Edition: Volume I

2 Contribution of the National Institute of Standards and Technology, 2010 Law EnforcementScientific AnalysisLegal Proceedings Police Agencies (local, state, federal) Forensic Laboratory Investigators/ Detectives CSI DNA Unit DNA Analysts Court System Prosecution Defense Judge Trial Legal framework and precedent Evidence submitted Scientific report(s) completed References submitted Validated scientific tests Laws and police training Conviction or exoneration Other Forensic Disciplines Research (introduces new methods) Evidence returned

3 Contribution of the National Institute of Standards and Technology, 2010 Sample Obtained from Crime Scene or Paternity Investigation DNA Extraction DNA Extraction DNA Quantitation DNA Quantitation PCR Amplification of Multiple STR markers PCR Amplification of Multiple STR markers Biology Separation and Detection of PCR Products (STR Alleles) Technology Sample Genotype Determination Genetics Comparison of Sample Genotype to Other Sample Results If match occurs, comparison of DNA profile to population databases Generation of Case Report with Probability of Random Match

4 Contribution of the National Institute of Standards and Technology, 2010 DNA Profile Comparison QK Court Database Search Exclusion (no match) Inclusion (match) May match another (K) Evidence (Question) sample Q Profile put on database Plea Report (with statistical weight) Q = K Q K Crime committed Biological material transferred Collection Extraction Quantitation STR Markers Data Interpretation Sample Storage Amplification Separation/ Detection Reference (Known) sample K Profile put on database Steps Involved Suspect developed Collection Extraction Quantitation STR Markers Data Interpretation Sample Storage Amplification Statistical Interpretation Characterization Separation/ Detection Biology Technology Genetics Serology Steps Involved QUALITYASSURANCEQUALITYASSURANCE QUALITYASSURANCEQUALITYASSURANCE May match another (K)

5 Contribution of the National Institute of Standards and Technology, end | Phosphate | SugarBase… | Phosphate | SugarBase… | 3end (a) (b) 5 3

6 Contribution of the National Institute of Standards and Technology, 2010 A = T G C T = A A = T C G T C C A G G T A G C T = A C G A = T G C denatured strands hybridized strands Hydrogen bonds C G G C Phosphate-sugar backbone

7 Contribution of the National Institute of Standards and Technology, 2010 The Human Genome X Y Sex- chromosomes Autosomes 3.2 billion bp Nuclear DNA - Located in cell nucleus 2 copies per cell mtDNA 16,569 bp mtDNA Located in mitochondria (multiple copies in cell cytoplasm) 100s of copies per cell Only single copy of each autosome shown

8 Contribution of the National Institute of Standards and Technology, 2010 p (short arm) centromere telomere q (long arm) telomere Band 5 Band 3 Chromosome 12 12p3 12q5

9 Contribution of the National Institute of Standards and Technology, repeats 3 repeats AGACTAGACATT AGATTAGGCATT (AATG)(AATG)(AATG) (AATG)(AATG) (B) Length polymorphism (A) Sequence polymorphism

10 Contribution of the National Institute of Standards and Technology, Homologous pair of chromosomes Locus A Locus B Allele 1 Allele 2 Allele 1

11 Contribution of the National Institute of Standards and Technology, X mtDNA Y Maternal Contribution Paternal Contribution X Y mtDNA Male Childs Full Genome Nuclear DNA Mitochondrial DNA Autosomes Sex chromosomes X or Sex chromosome Zygote (diploid) Sperm (haploid) Egg (haploid)

12 Contribution of the National Institute of Standards and Technology, 2010 AA A A a a Aa aA aa Freq (A) = p Freq (a) = q (p + q) 2 = p 2 + 2pq + q 2 Punnett square p2p2 qp pq q2q2 p q p q Father gametes (sperm) Mother gametes (egg) p + q = 1 Resulting genotype combinations and frequencies AA Aa p2p2 2pq aa q2q2

13 Contribution of the National Institute of Standards and Technology, 2010 AAaa Aa 1.0 Frequency of a allele (q) Frequency of A allele (p) Frequency of genotype in population p2p2 2pq q2q2

14 Contribution of the National Institute of Standards and Technology, , , 25 20,25 20,22 22,25 20,22 22,2520,2220, ,24 22, , , ,22 17 (a) (b) (c) 22,2522, ,2520, Fathers alleles Mothers alleles #3 #5 #4 #1 #2 22,2520,22 20,2520, Fathers alleles Mothers alleles #13 #14 #12 #7 #4

15 Contribution of the National Institute of Standards and Technology, 2010 Speed of Analysis (Technology) Power of Discrimination (Genetics) Low High SlowFast Markers Used (Biology) RFLP Single Locus Probes RFLP Multi-Locus Probes ABO blood groups Multiplex STRs DQ single STR D1S80 mtDNA PolyMarker

16 Contribution of the National Institute of Standards and Technology, 2010 ABABO A A or O A,B,AB, or O A,B, or AB A or O B A,B,AB, or O B or O A,B, or AB B or O AB A,B, or AB A or B O A or OB or OA or BO Fathers Blood Type Mothers Blood Type Childs Blood Type

17 Contribution of the National Institute of Standards and Technology, 2010 Multi-Locus Probe Probe 1Probe 2Probe 3 D1S7D2S44D4S139 Single-Locus Probe Probe 33.6

18 Contribution of the National Institute of Standards and Technology, 2010 probe Restriction site VNTR Small allele Large allele Small allele Large allele Sizing ladder 13 repeats 7 repeats Bands seen on autoradiogram of probed membrane probe

19 Contribution of the National Institute of Standards and Technology, 2010 TGCA GG CC TAACG ACGT CC GG ATTGC TGCA CTGCA G TAACG ACGT G ACGTC ATTGC TGCA G ANTC TAACG ACGT CTNA G ATTGC HaeIII HinfI PstI

20 Contribution of the National Institute of Standards and Technology, 2010 GTCCAGTCG PCR product (denatured) Biotin Strepavidin HRP TMB (colorless) Colored precipitate CAGGTCAGC Nylon membrane Immobilized SSO probe 5 3 hybridization matchno match View from above nylon membrane Allele 1Allele 2

21 Contribution of the National Institute of Standards and Technology, 2010 Nominal allele specific dots Control dot Subtype allele specific dots C All but DQA C All but AMPLITYPE TM DQ-Alpha SAB LDLR AB GYPA AB HBGG AB D7S8 AB GC C C S dot (A) (B) 1.2/3 AB BBABBC

22 Contribution of the National Institute of Standards and Technology, 2010 Allelic Ladder Allelic Ladder Positive Control Sample 1Sample

23 Contribution of the National Institute of Standards and Technology, 2010 Minisatellite Marker (D1S80) GAGGACCACCAGGAAG Repeat region Flanking regions 16 bp repeat unit STR Marker (TH01) TCAT Repeat region Flanking regions 4 bp repeat unit

24 Contribution of the National Institute of Standards and Technology, 2010 Allelic Ladders Sample 1 Sample CSF1PO TPOX TH Allelic Ladders 6 14

25 Contribution of the National Institute of Standards and Technology, TAAATGATTCC-5 ATT ATTTACTAA ATTTACT ATTTAC ATTT ATTTA AT ATTTACTA ATTTACTAAG ATTTACTAAGG A DNA template 53 Primer anneals Extension produces a series of ddNTP terminated products each one base different in length Each ddNTP is labeled with a different color fluorescent dye Sequence is read by noting peak color in electropherogram (possessing single base resolution)

26 Contribution of the National Institute of Standards and Technology, 2010

27

28

29 ORGANIC FTA Paper CHELEX Blood stain PUNC H WASH Multiple Times with extraction buffer PERFORM PCR PCR Reagent s SDS, DTT, EDTA and proteinase K INCUBATE (56 o C) Phenol, chloroform, isoamyl alcohol QUANTITATE DNA Apply blood to paper and allow stain to dry Blood stain VORTEX (NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES) Water INCUBATE (ambient) 5% Chelex INCUBATE (100 o C) REMOVE supernatant INCUBATE (56 o C) QUANTITATE DNA PERFORM PCR Centrifuge REMOVE supernatant TRANSFER aqueous (upper) phase to new tube CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge TE buffer

30 Contribution of the National Institute of Standards and Technology, 2010 Perpetrators sperm mixed with victims epithelial cells Centrifuge REMOVE supernatant SDS, EDTA and proteinase K (cell lysis buffer) Remove a portion of the mixed stain SDS, EDTA and proteinase K + DTT Incubate at 37 o C sperm pellet DTT lyses sperm heads Male Fraction Female Fraction sperm pellet Differential Extraction

31 Contribution of the National Institute of Standards and Technology, 2010 Too much DNA amplified (a)(b) Too little DNA amplified (c) Within optimal range

32 Contribution of the National Institute of Standards and Technology, ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng Calibration standards Unknown Samples 2.5 ng

33 Contribution of the National Institute of Standards and Technology, 2010 Polymerization and Strand Displacement R Q Forward primer Reverse primer Forward primer Reverse primer Q R 3 Probe Cleavage (release of reporter dye) Forward primer Reverse primer Q R Completion of Polymerization TaqMan probe Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity

34 Contribution of the National Institute of Standards and Technology, 2010

35 Cycle Number Normalized Fluorescence threshold CTCT Exponential product growth Linear product growth Plateau ΔRnΔRn Negative control ab cd e Standard curve CTCT Log[DNA] a b c d e N c = N o (1 + E) c If efficiency is close to 100% (E = 1), then the product copy number (N c ) doubles the target copy number (N o ) with each cycle (c).

36 Contribution of the National Institute of Standards and Technology, o C 60 o C 72 o C Time Temperature Single Cycle Typically cycles performed during PCR 94 o C 60 o C 72 o C The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a hot-start PCR

37 Contribution of the National Institute of Standards and Technology, 2010 Separate strands (denature) Add primers (anneal) Make copies (extend primers) Repeat Cycle, Copying DNA Exponentially Starting DNA Template Forward primer Reverse primer

38 Contribution of the National Institute of Standards and Technology, 2010

39 (a) (b)

40 Contribution of the National Institute of Standards and Technology, 2010 (a) Simultaneous amplification of three locations on a DNA template Locus A Locus C Locus B (b) Resolution of PCR products with size-based separation method A C B small large

41 Contribution of the National Institute of Standards and Technology, 2010 STR repeat region GATA PCR product size generated DNA template containing STR marker Reverse PCR primer Forward PCR primer Fluorescent dye (a) (b) Flanking regions 4 repeat units 5 repeat units 6 repeat units

42 Contribution of the National Institute of Standards and Technology, TTTCCC TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3 3-AAAGGG AGTA AGTA AGTA AGTA AGTA AGTA AGTGGTACCT

43 Contribution of the National Institute of Standards and Technology, 2010 Combine and re-amplify Allelic Ladder Individual Samples

44 Contribution of the National Institute of Standards and Technology, 2010 AmpFlSTR Identifiler kit (Applied Biosystems) 6-FAM (Blue) VIC (Green) NED (Yellow) PET (Red) D8S1179 D21S11 D7S820CSF1PO D3S1358TH01D13S317D16S539D2S1338 D19S433D18S51 TPOX VWA AMELD5S818FGA GS500 LIZ size standard LIZ (Orange) D3S1358 TH01 D21S11D18S51Penta E D5S818D13S317D7S820D16S539CSF1PO Penta D AMELVWAD8S1179 TPOX FGA PowerPlex 16 kit (Promega Corporation) ILS600 CXR size standard CXR (Red) FL (Blue) JOE (Green) TMR (Yellow) PCR product size (bp)

45 Contribution of the National Institute of Standards and Technology, 2010 D3S1358 TH01D21S11 D18S5 1 Penta E D5S818 D13S317 D7S820 D16S539 Penta D CSF1PO Amelogenin (sex-typing) VWAD8S1179 TPOX FGA blue panel Overlay of all 4 colors (including internal size standard) yellow panel green panel red panel ILS600 DNA sizing standard 200 bp 300 bp100 bp 400 bp500 bp

46 Contribution of the National Institute of Standards and Technology, 2010 D3S1358 (8 alleles) VWA (14 alleles) D16S539 (9 alleles) D2S1338 (14 alleles) Blue panel Green panel Yellow panel Orange panel D21S11 (24 alleles) D8S1179 (12 alleles) D18S51 (23 alleles) TH01 (10 alleles) FGA low (19 alleles) FGA high (9 alleles) 250 bp* 139bp 200 bp 160 bp 300 bp 340 bp 350 bp 150 bp LIZ-labeled GS500 DNA sizing standard 100 bp Red panel D19S433 (15 alleles) D5S818 (10 alleles) TPOX (8 alleles) D13S317 (8 alleles) D7S820 (10 alleles) CSF1PO (10 alleles) AMEL (2 alleles)

47 Contribution of the National Institute of Standards and Technology, 2010 Fluorescent dye at 5end Non-nucleotide linkers (mobility modifiers) Primer sequence PCR amplification generates a labeled PCR product containing the mobility modifiers 5-end 3-end For each linker unit added, there is an apparent migration shift of 2.5 bp

48 Contribution of the National Institute of Standards and Technology, 2010 D7S820 CSF1PO NED-labeled (yellow) JOE-labeled (green) D7S820 CSF1PO FAM-labeled (blue) (a) COfiler kit allele relative size ranges (b) Identifiler kit allele relative size ranges bp bp bp bp bp bp bp bp Size overlap 10 non-nucleotide linkers = +25 bp shift

49 Contribution of the National Institute of Standards and Technology, 2010 CSF1PO forward primer CSF1PO reverse primer (AGAT) 6-15 (a) PowerPlex 1.1 Kit 91 bp 128 bp TMR-labeled PCR product sizes = bp CSF1PO forward primer CSF1PO reverse primer (b) PowerPlex 16 Kit 13 bp 238 bp JOE-labeled (AGAT) 6-15 PCR product sizes = bp +30 bp shift in size

50 Contribution of the National Institute of Standards and Technology, 2010 X Y 6 bp deletion Female: X, X Male: X, Y 1:1 Mixture: 3X + 1Y X = 212 bp Y = 218 bp X = 106 bp Y = 112 bp AmpFlSTR kits and PowerPlex 16 PowerPlex 1.1

51 Contribution of the National Institute of Standards and Technology, 2010

52 - Voltage Gel Loading well + anode cathode Side view Top view Gel lanes DNA bands Buffer + - Gel stand

53 Contribution of the National Institute of Standards and Technology, 2010 Laser Inlet Buffer Capillary filled with polymer solution 5-20 kV -+ Outlet Buffer Sample tray Detection window (cathode) (anode) Data Acquisition Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession

54 Contribution of the National Institute of Standards and Technology, 2010 Mixture of dye- labeled PCR products from multiplex PCR reaction CCD Panel (with virtual filters) Argon ion LASER (488 nm) Color Separation Fluorescenc e ABI Prism spectrograph Size Separation Processing with GeneScan/Genotyper software Sample Interpretation Sample Injection Sample Separation Sample Detection Sample Preparatio n Capillary (filled with polymer solution)

55 Contribution of the National Institute of Standards and Technology, 2010 (a) Larger DNA molecules interact more frequently with the gel and are thus retarded in their migration through the gel Gel (b) Ogston SievingReptation Small DNA molecules Long DNA molecules Gel

56 Contribution of the National Institute of Standards and Technology, 2010 h ex h em SoSo S1S1 S1S1 energy (a) Excitation Emission Wavelength (nm) 1 3 ex max em max Fluorescence (b) Stokes shift

57 Contribution of the National Institute of Standards and Technology, 2010 Fluorescent dNTPs are incorporated into both strands of PCR product Ethidium bromide DNA labeled with intercalating dye Unlabeled DNA SYBR Green Intercalator inserts between base pairs on double-stranded DNA One strand of PCR product is labeled with fluorescent dye Fluorescent dye labeled primer (a) (b) (c)

58 Contribution of the National Institute of Standards and Technology, 2010 FAM (blue) JOE (green) TAMRA (yellow) ROX (red)

59 Contribution of the National Institute of Standards and Technology, WAVELENGTH (nm) Filter Set F with color contributions 5-FAMJOENED ROX Laser excitation (488 nm, nm) Laser excitation (488 nm, nm) Normalized Fluorescent Intensity

60 Contribution of the National Institute of Standards and Technology, 2010 Scan number Relative Fluorescence Units DNA size in base pairs Relative Fluorescence Units Region shown below (a) (b)

61 Contribution of the National Institute of Standards and Technology, 2010 Capillary Heat plate Detectio n window electrode Autosampler Gel block Syringe (with polymer) Outlet buffer reservoir Inlet buffer reservoir Sample tray Samples

62 Contribution of the National Institute of Standards and Technology, 2010 Mechanical pump (with polymer) Capillary array Oven Detectio n window electrodes Autosampler Lower gel block Polyme r bottle Outlet buffer reservoir Inlet buffer reservoir Sample tray Fan

63 Contribution of the National Institute of Standards and Technology, 2010 Capillaries Electrodes for Injection

64 Contribution of the National Institute of Standards and Technology, 2010 Data Collection Peak Identification Data Review by Analyst/Examiner Color Separation Peak Sizing Comparison to Allelic Ladder Confirmation of Results by Second Analyst/Examiner Genotype Assignment to Alleles GeneScan software Genotyper software Internal size standard Matrix file (spectral calibration) Allelic ladder sample GeneMapperID software Expert Systems (e.g., FSS-i3, TrueAllele) Peak Editing to Remove Artifact Calls User-defined thresholds

65 Contribution of the National Institute of Standards and Technology, RFUs 150 RFUs Analytical Threshold Interpretation Threshold Baseline Noise Peak reliable, but only used for exclusions Peak reliable, can be used for inclusions Peak not considered reliable Values shown for example purposes only (should be based empirically on a labs internal validation)

66 Contribution of the National Institute of Standards and Technology, 2010 DNA fragment peaks in sample DNA Size Data Point bp bp DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard (a) (b) Time (minutes)

67 Contribution of the National Institute of Standards and Technology, 2010 Allelic ladderPCR-amplified sample Internal size standard Color-separated and sized allele peaks for each locus Data from CE instrument (prior to color separation and peak sizing) Genotyping performed by comparing allelic ladder to sample results Color separation and peak sizing Locus 1 Genotype = 12, 14 All ladder alleles sized using internal size standard All sample alleles sized using internal size standard Genotyping allele bins (+/-0.5 bp around ladder allele) Alleles (# repeats)

68 Contribution of the National Institute of Standards and Technology, 2010 Dye blob STR alleles stutter Pull-up (bleed-through) spike Blue channel Green channel Yellow channel Red channel D3S1358 Stutter products 6.0%7.8% Incomplete adenylation D8S1179 -A +A -A +A Biological (PCR) artifacts

69 Contribution of the National Institute of Standards and Technology, 2010 (a)(b)(c) Allelic ladder

70 Contribution of the National Institute of Standards and Technology, = S 25 -L 25 = = bp 2 = S OL - L 28 = = bp c = | | = | | = 0.99 bp

71 Contribution of the National Institute of Standards and Technology, 2010 (a)(b) Type 1Type 2

72 Contribution of the National Institute of Standards and Technology, 2010 * * Allele 6 amplicon has dropped out Imbalance in allele peak heights Heterozygous alleles are well balanced No mutation Mutation at 3-end of primer binding site (allele dropout) Mutation in middle of primer binding site (a) (b) (c)

73 Contribution of the National Institute of Standards and Technology, 2010 DNA Profile (with specific alleles) Rarity estimate of DNA profile (e.g., RMP or LR) Genetic formulas Population allele frequencies

74 Contribution of the National Institute of Standards and Technology, 2010 Decide on Number of Samples and Ethnic/Racial Grouping Gather Samples Analyze Samples at Desired Genetic Loci Summarize DNA types Ethnic/ Racial Group 1 Ethnic/ Racial Group 2 Determine Allele Frequencies for Each Locus Perform Statistical Tests on Data Hardy-Weinberg equilibrium for allele independence Linkage equilibrium for locus independence Usually >100 per group Use Database(s) to Estimate an Observed DNA Profile Frequency Often anonymous samples from a blood bank See Table 11.1 Examination of genetic distance between populations

75 Contribution of the National Institute of Standards and Technology, 2010 Paternal Allele Maternal Allele Genotype Locus 1 DNA Profile Paternal Allele Maternal Allele Genotype Locus 2 Paternal Allele Maternal Allele Genotype Locus 3 HWE Linkage Equilibrium (product rule)

76 Contribution of the National Institute of Standards and Technology, 2010 National Level NDIS (FBI Laboratory) SDIS (Richmond, Virginia) SDIS (Tallahassee, Florida) LDIS (Tampa) LDIS (Orlando) LDIS (Broward County) LDIS (Roanoke) LDIS (Norfolk) LDIS (Fairfax) State Level Local Level

77 Contribution of the National Institute of Standards and Technology, 2010 Convicted Offender Index Offenders (N)Crime Samples (C) Forensic Index Arrestee Index Arrestees (A) Offender Hit Forensic Hit

78 Contribution of the National Institute of Standards and Technology, 2010 Degraded DNA sample D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D Agarose yield gel results Smear of degraded DNA fragments High relative molecular mass DNA in a tight band (a) (b) Good quality DNA Degraded DNA

79 Contribution of the National Institute of Standards and Technology, 2010 Full Profile (Good Quality) Partial Profile (Poor Quality) (a) (b) DNA size (bp) relative to an internal size standard (not shown) Relative fluorescence units (RFUs)

80 Contribution of the National Institute of Standards and Technology, 2010 STR repeat region miniSTR primer Conventional PCR primer (a) (b) Conventional STR test (COfiler kit) MiniSTR assay (using Butler et al primers) 150 bp smaller

81 Contribution of the National Institute of Standards and Technology, 2010 (a) Single Source D3S1358TH01D13S317D16S539D2S ,169,9.38,129,917,19 (b) Mixed Source D3S1358TH01D13S317D16S539D2S1338

82 Contribution of the National Institute of Standards and Technology, 2010 Type AType BType C

83 Contribution of the National Institute of Standards and Technology, 2010 >2 alleles at a locus, except tri- allelics? Single Source DNA Sample NO Mixed DNA Sample YES Differentiate a Major/Minor Component? Determine STR profile and compute RMP YES Is the sample a mixture? TYPE B NO YES Stochastic Effects ? Possible Low Level DNA) ? YES Assume # Contributor s? TYPE C TYPE A NO A biostatistical analysis must be performed Probability of Exclusion [CPE] RMNE Likelihood Ratio [LR] YES NO Are # of contributors defined? A biostatistical analysis should not be performed Determine component profile(s) and compute RMP for major

84 Contribution of the National Institute of Standards and Technology, 2010 Identify the Presence of a Mixture Consider All Possible Genotype Combinations Estimate the Relative Ratio of the Individuals Contributing to the Mixture Identify the Number of Potential Contributors Designate Allele Peaks Compare Reference Samples Step #1 Step #2 Step #3 Step #4 Step #5 Step #6

85 Contribution of the National Institute of Standards and Technology, pg 50 pg 10 pg Allele dropout Severe imbalance Good heterozygote peak balance

86 Contribution of the National Institute of Standards and Technology, 2010

87 Two possible alleles Human Alu Repeat (~300 bp) AluI 400 bp 100 bp long (+) allele short (-) allele

88 Contribution of the National Institute of Standards and Technology, 2010 Autosomal (passed on in part, from all ancestors) Y-Chromosome (passed on complete, but only by sons) Mitochondrial (passed on complete, but only by daughters) Lineage Markers

89 Contribution of the National Institute of Standards and Technology, 2010 Female-Male Mixture Performance with Autosomal vs. Y-Chromosome DNA Markers Female Victim DNA Profile Male Perpetrator DNA Profile DNA Profile from Crime Scene Autosomal STR Profile Y-Chromosome STR Profile No signal observed

90 Contribution of the National Institute of Standards and Technology, 2010 ? uncle 3 rd cousin (paternal)

91 Contribution of the National Institute of Standards and Technology, 2010

92 Control region (D-loop) 1/16,569 cyt b HV1 HV mtGenome 16,569 bp 1 Heavy (H) strand Light (L) strand Coding region HV

93 Contribution of the National Institute of Standards and Technology, MtDNA Haplotype Groups: 1 2,3,6,8,11,13,15,16 4,9, ,17,18 MtDNA Haplotype Groups: 1 2,3,6,8,11,13,15,16 4,9, ,17,18 A B B C C C D B B B B B B E F G G G

94 Contribution of the National Institute of Standards and Technology, 2010 Compare with database to determine haplotype frequency Extract mtDNA from evidence (Q) sample PCR Amplify HV1 and HV2 Regions Sequence HV1 and HV2 Amplicons (both strands) Confirm sequence with forward and reverse strands Note differences from Anderson (reference) sequence Compare Q and K sequences Performed separately and preferably after evidence is completed Extract mtDNA from reference (K) sample PCR Amplify HV1 and HV2 Regions Sequence HV1 and HV2 Amplicons (both strands) Confirm sequence with forward and reverse strands Note differences from Anderson (reference) sequence

95 Contribution of the National Institute of Standards and Technology, 2010 Sample Q 16093C 16129A Sample K 16093C 16129A ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT rCRS ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT Q K (a) mtDNA Sequences Aligned with rCRS (positions ) (b) Reporting Format with Differences from rCRS

96 Contribution of the National Institute of Standards and Technology, 2010 Thomas Jefferson II Field JeffersonPeter Jefferson President Thomas Jefferson Eston Hemings Same Y Haplotype Jefferson Y Haplotype Jefferson Y Haplotype ? Randolph Jefferson

97 Contribution of the National Institute of Standards and Technology, 2010 Tsarina Alexandra Tsar Nicholas II Xenia Cheremeteff- Sfiri Prince Philip Duke of Edinburgh Prince Philip Duke of Edinburgh Georgij Romanov Georgij Romanov Mitotype 16111T 16357C 263G 315.1C Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.1C 16169T/C Louise of Hesse-Cassel

98 Contribution of the National Institute of Standards and Technology, ,14 8,12 12,14 11,128,1412,148,11 Obligate paternal allele C,D A,B B,C mother father child (b) Example (a) Mendelian Inheritance

99 Contribution of the National Institute of Standards and Technology, 2010 child Mother (known parent) Alleged father ? Rules of Inheritance 1)Child has two alleles for each autosomal marker (one from mother and one from biological father) 2)Child will have mothers mitochondrial DNA haplotype (barring mutation) 3)Child, if a son, will have fathers Y- chromosome haplotype (barring mutation) Random man Missing child Alleged mother Alleged father ? Parentage (Paternity) Testing Reverse Parentage Testing (Missing Persons Investigation) (a) (b)

100 Contribution of the National Institute of Standards and Technology, 2010 DNA profile from mass disaster victim DNA profile from direct reference (toothbrush believed to have belonged to the victim) (a) Direct comparison (b) Kinship analysis D5S818D13S317 D7S820 D16S539 CSF1PO Penta D ? son wife victim D5S818D13S317D7S820 D16S539CSF1POPenta D 10,109,109,138,98,1411,13 son wife 10,128,108,98,12 11,13 10,109,1211,139,911,1412,13 ?,109,??,13 9,? ?,14 11,? or ?,13 victim (father) actual profile Predicted victim profile mass disaster victim profile

101 Contribution of the National Institute of Standards and Technology, ,18 (a)(b) 15,18 15,1714,18 13,17 15,17

102 Contribution of the National Institute of Standards and Technology, 2010 Core set of markers (e.g., CODIS 13 STRs) Past and Present Future (a) (b) (c) (d)

103 Contribution of the National Institute of Standards and Technology, 2010 Loci Described Use in Casework Court Presentation/ Acceptance Internal Validation Research Government Funded or Private Development Typically by Commercial Manufacturer Forensic Application Forensic Labs Assay Constructed Population Study Information Gathered Released to Community Kit Developed Kit Tested

104 Contribution of the National Institute of Standards and Technology, 2010 Lower amounts of DNA being tested Challenging kinship search questions Standard STR Typing (DNA Profile) Core Competency Sufficient DNA quantity (ng) Direct Matching (or Parentage) Solution: Replicate Testing Solution: Additional Markers (Y-chromosome, more STRs) and Multiple Reference Samples Touch DNA Attempts (poor quality, mixtures, low-level stochastic effects) Familial Searching Attempts (fishing for brothers or other relatives)

105 Contribution of the National Institute of Standards and Technology, ,12 8,13 12,138,9 9,12 9,13 Q (query) K (database) (b) 9,139,12 8,138, Fathers alleles Mothers alleles (a)

106 Contribution of the National Institute of Standards and Technology, 2010 Set up two hypotheses (H 0 and H 1 ) Collect data and calculate the test statistic (S) Look up the critical value (C) and define the region of rejection for the test statistic Is S C? yes Accept H 0 (Reject H 1 ) Accept H 1 (Reject H 0 ) no Select appropriate statistical model Specify the level of significance and its critical value (C)

107 Contribution of the National Institute of Standards and Technology, 2010 Truth about the population Decision based on sample examined Correct decision Type II error Type I errorCorrect decision H 0 True H 0 False Accept H 0 Reject H 0 (Accept H 1 ) Correct decision Wrongfully acquitted Wrongfully accused Correct decision (b) Example Not Guilty Guilty Courtroom Verdict Saint Sinner Defendant (a) Hypothesis Testing Decisions


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