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Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler Lineage Markers (Y/mtDNA)
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Chapter Summary Unlike autosomal DNA markers (e.g, the 13 CODIS STRs) that are inherited from both parents and shuffled with each generation, lineage markers are uniparentally-passed from one generation to the next without changing (except for mutational events). Paternal lineages may be tracked with genetic markers on the non-recombining portion of the Y-chromosome that are passed from father to son. Maternal lineages may be followed as mothers pass their mitochondrial DNA (mtDNA) on to their children. X-chromosome markers may also be helpful in specific situations. Genetic genealogy companies use many of the same Y-STR markers and regions of mtDNA to trace ancestry of avid genealogists. The primary advantage of Y-STRs in a forensic setting is to enable male-specific amplification from sexual assault evidence containing a mixture of male and female cells that cannot be separated by differential extraction, such as fingernail scrapings. Mitochondrial DNA testing, which is not as informative as autosomal DNA analysis, is in many cases a last resort with difficult evidentiary samples such as bones, teeth, or hair. Success rates improve with mtDNA analysis because mtDNA is present in hundreds of copies per cell. MtDNA results are more time consuming and labor intensive to generate (and more prone to error) than STR typing making it more challenging to perform routinely. Thus, there are a limited number of mtDNA labs compared to STR typing facilities.
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Role of Y-STRs and mtDNA Compared to Autosomal STRs Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shafts
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Value of Y-Chromosome Markers Application Advantage Forensic casework on sexual assault evidence Male-specific amplification (can avoid differential extraction to separate sperm and epithelial cells) Paternity testing Male children can be tied to fathers in motherless paternity cases Missing persons investigations Patrilineal male relatives may be used for reference samples Human migration and evolutionary studies Lack of recombination enables comparison of male individuals separated by large periods of time Historical and genealogical research Surnames usually retained by males; can make links where paper trail is limited J.M. Butler (2005) Forensic DNA Typing, 2nd Edition; Table 9.1
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Lineage Markers: Y-STRs and mtDNA Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred
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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 John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.1 Patterns of Genetic Inheritance
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(A) (B) Pseudoautosomal Region 1 (PAR1) Pseudoautosomal Region 2 (PAR2) Non-Recombining Portion of Y Chromosome (NRY) Male-specific region of the Y (MSY) 50 Mb X Y 154 Mb Yp Yq Heterochromatic region (not sequenced) ~30 Mb Euchromatic region (23 Mb) centromere recombination recombination Comparison of X and Y Chromosomes
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DYS385 a/b a = b a  b DYS389 I/II (A) (B) I II F primer F primer R primer a b Duplicated regions are 40,775 bp apart and facing away from each other F primer R primer F primer R primer DYS389I DYS389II Multi-Copy (Duplicated) Marker Single Region but Two PCR Products (because forward primers bind twice)
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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 John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.2
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? uncle 3rd cousin (paternal) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.3 Patrilineal Inheritance Where All Shaded Males Have the Same Y-Chromosome (barring any mutations) Y-STRs permit extension of possible reference samples in missing persons cases
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John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.4 Y-STR Result Using Yfiler Kit
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Control region (D-loop) 1/16,569 cyt b HV1 HV2 16024 16365 73 340 16024 576 mtGenome ‘16,569’ bp 1 Heavy (H) strand Light (L) strand Coding region HV3 438 574 John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.5 Mitochondrial DNA Genome and Regions Commonly Analyzed
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Control region (D-loop) 1/16,569 cyt b ND5 ND6 ND4 ND4L ND3 COIII ATP6 ATP8 COII 12S rRNA 16S rRNA ND1 ND2 COI OH 9-bp deletion OL F V L1 I Q M W A N C Y S1 D K G R H S2 L2 E P T HV1 HV2 16024 16365 73 340 16024 576 “16,569” bp 1 22 tRNAs 2 rRNAs 13 genes Heavy (H) strand Light (L) strand
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HV1 HV2 HV3 16024 16365 73 340 438 574 16519 16569/1 342 bp 268 bp 137 bp F15989 R16251 F16190 R16410 VR1 VR2 C-stretch AFDIL “mini-primer” set PSI (263 bp) PSII (221 bp) F15 R285 F155 R381 PSIII (271 bp) PSIV (227 bp) C-stretch MPS1A (170 bp) MPS1B (126 bp) MPS2A (133 bp) MPS2B (143 bp) MPS3A (126 bp) MPS3B (132 bp) MPS4A (142 bp) MPS4B (158 bp) AFDIL primer set
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GAAAAAGTCT TTAACTCCAC CATTAGCACC CAAAGCTAAG ATTCTAATTT AAACTATTCT CTTTTTCAGA AATTGAGGTG GTAATCGTGG GTTTCGATTC TAAGATTAAA TTTGATAAGA 15970 15980 15990 16000 16010 16020 CTGTTCTTTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACA GACAAGAAAG TACCCCTTCG TCTAAACCCA TGGTGGGTTC ATAACTGAGT GGGTAGTTGT 16030 16040 16050 16060 16070 16080 C C A ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT TGGCGATACA TAAAGCATGT AATGACGGTC GGTGGTACTT ATAACATGCC ATGGTATTTA 16090 16100 16110 16120 16130 16140 ACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTA TGAACTGGTG GACATCATGT ATTTTTGGGT TAGGTGTAGT TTTGGGGGAG GGGTACGAAT 16150 16160 16170 16180 16190 16200 CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACC GTTCGTTCAT GTCGTTAGTT GGGAGTTGAT AGTGTGTAGT TGACGTTGAG GTTTCGGTGG 16210 16220 16230 16240 16250 16260 T T C G C CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGC GGAGTGGGTG ATCCTATGGT TGTTTGGATG GGTGGGAATT GTCATGTATC ATGTATTTCG 16270 16280 16290 16300 16310 16320 C CATTTACCGT ACATAGCACA TTACAGTCAA ATCCCTTCTC GTCCCCATGG ATGACCCCCC GTAAATGGCA TGTATCGTGT AATGTCAGTT TAGGGAAGAG CAGGGGTACC TACTGGGGGG 16330 16340 16350 16360 16370 16380 TCAGATAGGG GTCCCTTGAC CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCT AGTCTATCCC CAGGGAACTG GTGGTAGGAG GCACTTTAGT TATAGGGCGT GTTCTCACGA 16390 16400 16410 16420 16430 16440 FBI A1 (L15997) Roche (F15975) HV1 16093 16126 16129 HVI C-stretch Roche IA Roche ID Roche IC Roche IE HV1 FBI B1 (H16391) Roche (R16418) Hypervariable Region I 16024-16365 342 bp examined SSO Probes 16093 16126 16129 16270 16278 16304 16309 16311 16362 Only 9 sites examined
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GATCACAGGT CTATCACCCT ATTAACCACT CACGGGAGCT CTCCATGCAT TTGGTATTTT CTAGTGTCCA GATAGTGGGA TAATTGGTGA GTGCCCTCGA GAGGTACGTA AACCATAAAA 10 20 30 40 50 60 G CGTCTGGGGG GTATGCACGC GATAGCATTG CGAGACGCTG GAGCCGGAGC ACCCTATGTC GCAGACCCCC CATACGTGCG CTATCGTAAC GCTCTGCGAC CTCGGCCTCG TGGGATACAG 70 80 90 100 110 120 C T C GCAGTATCTG TCTTTGATTC CTGCCTCATC CTATTATTTA TCGCACCTAC GTTCAATATT CGTCATAGAC AGAAACTAAG GACGGAGTAG GATAATAAAT AGCGTGGATG CAAGTTATAA 130 140 150 160 170 180 G C T G ACAGGCGAAC ATACTTACTA AAGTGTGTTA ATTAATTAAT GCTTGTAGGA CATAATAATA TGTCCGCTTG TATGAATGAT TTCACACAAT TAATTAATTA CGAACATCCT GTATTATTAT 190 200 210 220 230 240 A ACAATTGAAT GTCTGCACAG CCACTTTCCA CACAGACATC ATAACAAAAA ATTTCCACCA TGTTAACTTA CAGACGTGTC GGTGAAAGGT GTGTCTGTAG TATTGTTTTT TAAAGGTGGT 250 260 270 280 290 300 AACCCCCCCT CCCCCGCTTC TGGCCACAGC ACTTAAACAC ATCTCTGCCA AACCCCAAAA TTGGGGGGGA GGGGGCGAAG ACCGGTGTCG TGAATTTGTG TAGAGACGGT TTGGGGTTTT 310 320 330 340 350 360 ACAAAGAACC CTAACACCAG CCTAACCAGA TTTCAAATTT TATCTTTTGG CGGTATGCAC TGTTTCTTGG GATTGTGGTC GGATTGGTCT AAAGTTTAAA ATAGAAAACC GCCATACGTG 370 380 390 400 410 420 TTTTAACAGT CACCCCCCAA CTAACACATT ATTTTCCCCT CCCACTCCCA TACTACTAAT AAAATTGTCA GTGGGGGGTT GATTGTGTAA TAAAAGGGGA GGGTGAGGGT ATGATGATTA 430 440 450 460 470 480 HV2 73 146 150 HV2 C-stretch Roche IIA Roche IID Roche IIB Roche IIC HV2 FBI D1 (H408) Roche (RR429) FBI C1 (L048) Roche (F15) 195 189 152 247 200 198 Hypervariable Region II 73-340 268 bp examined SSO Probes 73 146 150 152 189 195 198 200 247 Only 9 sites examined
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1 2 3 5 4 12 11 10 9 6 7 8 18 17 15 16 13 14 MtDNA Haplotype Groups: 1 2,3,6,8,11,13,15,16 4,9,10 5 7 12 14,17,18 A B B C C C D B B B B B B E F G G G John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.6 Mitochondrial DNA Inheritance Patterns
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1 2 3 5 4 12 11 10 9 6 7 8 18 17 15 16 13 14 MtDNA Haplotype Groups: 1 2,3,6,8,11,13,15,16 4,9,10 5 7 12 14,17,18 A B B C C C D B B B B B B E F G G G Figure 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Mitochondrial DNA Inheritance Sons receive mtDNA from their mothers but do not pass it on to their offspring
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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 John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.7 Process for Evaluating mtDNA Samples
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Sample Q 16093C 16129A Sample K 16093C 16129A ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT 16090 16100 16110 16120 16130 16140 rCRS ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT Q K ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT (a) mtDNA Sequences Aligned with rCRS (positions 16071-16140) (b) Reporting Format with Differences from rCRS John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 16.8 Comparison of mtDNA Sequences Q = question sample (usually evidence) K = known reference sample
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Thomas Jefferson II Field Jefferson Peter Jefferson President Thomas Jefferson Eston Hemings Same Y Haplotype Jefferson Y Haplotype Jefferson Y Haplotype ? Randolph Jefferson John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 16.1 Ancestry of Thomas Jefferson and Eston Hemings Male Lines
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Thomas Jefferson II Field Jefferson Peter Jefferson President Thomas Jefferson Eston Hemings Thomas Woodson Different Y Haplotype Same Y Haplotype Jefferson Y Haplotype Jefferson Y Haplotype ? Historical Investigation DNA Study
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Tsarina Alexandra Tsar Nicholas II Xenia Cheremeteff-Sfiri Prince Philip Duke of Edinburgh Georgij Romanov Mitotype 16111T 16357C 263G 315.1C Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.1C 16169T/C 16169T/C Louise of Hesse-Cassel John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 16.5 Lineage of Romanov Family
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The Romanovs (Last Russian Czar) Tsarina Alexandra Tsar Nicholas II Xenia Cheremeteff-Sfiri Prince Philip Duke of Edinburgh Georgij Romanov Mitotype 16111T 16357C 263G 315.1C Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.1C 16169T/C 16169T/C Louise of Hesse-Cassel SOURCES: Gill et al. (1994) Nature Genetics, 6, 130‑135.; Ivanov et al. (1996) Nature Genetics, 12, 417‑420; Stone, R. (2004) Science, 303, 753. D.N.A. Box 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
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Genghis Khan’s Y-Chromosome Profile Zerjal et al. (2003) Am. J. Hum. Genet. 72:717–721 http://www.ysearch.org
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3’-TAAATGATTCC-5’ ATT ATTTACTAA ATTTACT ATTTAC ATTT ATTTA AT ATTTACTA ATTTACTAAG ATTTACTAAGG A DNA template 5’ 3’ 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)
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HV1 C-stretch 16189T Poor quality sequence (two length variants out of phase) Good quality sequence Primer strategies typically used with C-stretch containing samples (A) (B) (C) C-stretch C-stretch Use of internal primers Double reactions from the same strand Figure 10.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Polymeric C-stretch in mtDNA Causes Problems with DNA Sequencing
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16093 (C/T) 16086 16101 Sequence Heteroplasmy at mtDNA position 16093
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(B) Reported Types K: 1-1-1-1-1-1-1-1-1-1 Q: 1-2-3-2-0-1-4-2-2-w1 IA 1 2 IC ID IE IIA IIB IIC IID 189 16093 HVI HVII 1 2 1 2 3 4 1 2 1 2 1 2 1 2 3 4 5 6 1 2 4 5 1 2 1 2 3 3 Ref 7 K Q (A) “blank” Comparison of Results with Roche LINEAR ARRAY
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Y-STR Loci in Use for Genetic Genealogy Family Tree DNA (12, 37, or 67 loci) – DYS19, DYS385 a/b, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS426, DYS439, DYS437, DYS447, DYS448, DYS449, DYS454, DYS455, DYS458, DYS459 a/b, DYS464 a/b/c/d, DYS438, DYS442, DYS460, GATA-H4, YCA II a/b, DYS456, DYS570, DYS576, DYS607, DYS724 a/b (CDY a/b), DYF395S1a/b, DYF406S1, DYS413 a/b, DYS425, DYS436, DYS444, DYS446, DYS450, DYS472, DYS481, DYS487, DYS490, DYS492, DYS511, DYS520, DYS531, DYS534, DYS537, DYS557, DYS565, DYS568, DYS572, DYS578, DYS590, DYS594, DYS617, DYS640, DYS641 Relative Genetics (18, 26, or 43 loci) – DYS19, DYS385 a/b, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS426, DYS438, DYS439, DYS447, DYS448, DYS460, YCAII a/b, GATA-H4, DYS393, DYS437, DYS454, DYS455, DYS461, DYS462, GGAAT1B07, GATA-A10, DYS635, DYS441, DYS442, DYS444, DYS445, DYS446, DYS449, DYS452, DYS456, DYS458, DYS459 a/b, DYS463, DYS464 a/b/c/d Ethnoancestry (18 loci) –DYS481, DYS487, DYS490, DYS494, DYS505, DYS522, DYS531, DYS533, DYS549, DYS556, DYS575, DYS578, DYS589, DYS594, DYS636, DYS638, DYS641, DYF406S1 + Y-SNPs Oxford Ancestors (10 loci) – DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS425, DYS426 GeoGene (6 loci) – DYS19, DYS388, DYS390, DYS391, DYS392, DYS393 + Y-SNPs
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More Y-STRs Provides Greater Confidence in Matching Results Between Potential Relatives
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www.Ysearch.org
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www.mitosearch.org
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9-14 [GATA] [GATA] 16-21 1 2 9-14 3 [GATA] [GATA] 1 [GATA] N4 N14 N3 N7 [GATA] [ATCT] 18-23 3 2 9-14 2 [AGAT] [AGAT] 2 [AGAT] N20 N3 N14 N10 [GATA] (A) (B) (C) (D) ATCTATCTTGAATTAATAGATTCAAGGTGATAGATATACAGATAGATAGATACATAGGTGGAGACAGATAGATGATAAATAGAAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATA Multiple Published Allele Nomenclatures for DYS439
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M42 M60 RPS4Y (M130) M168 M89 M45 M201 M52 M170 M172 A B C D E E3a F* G H I J2 K* L M N M175 M5 M11 P36 O Q R R1b M9 P* M91 YAP M174 M2 M96 12f2 J LLY22g M207 M173 P25 M269 R1b3 R1 This Y-SNP tree has been updated with additional markers and haplogroups… Y Chromosome Consortium 2002 Tree with 18 major haplogroups (A-R)
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Genetic Markers Examined with Ancestry Testing Y chromosome Passed from father to son without change (unless a mutation occurs) Two types of genetic markers are typically examined: Y-STRs (short tandem repeats) – provides finer detail Y-SNPs (single nucleotide polymorphisms) – big picture view mtDNA Passed from mother to offspring without change (unless a mutation occurs) Typically a section of the 16,569 bp mtDNA is sequenced – a haplogroup is assigned by presence of specific DNA mutations
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Chapter 16 – Points for Discussion Describe how lineage markers can be helpful in forensic DNA testing. In genetic genealogy testing? Will the availability and use of Y chromosome tests change the strategy for examining forensic DNA evidence in the future? If so, how? Discuss a casework situation in which Y-STR testing would be helpful. What are some of the challenges with trying to determine a reliable Y-STR profile frequency estimate when a suspect cannot be excluded from contributing to a crime scene sample? What parts of the Y-chromosome are recombine with the X-chromosome? What is the revised Cambridge reference sequence and why is it used? How is mtDNA heteroplasmy detected and why does it complicate mtDNA analysis?