Molecular Analysis of the Human Mitochondrial DNA Control Region for Forensic Identity Testing

Mitchell M. Holland1

1 Pennsylvania State University, University Park, Pennsylvania
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 14.7
DOI:  10.1002/0471142905.hg1407s74
Online Posting Date:  July, 2012
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Abstract

This unit highlights methods used to perform PCR amplification and sequence analysis of mitochondrial DNA (mtDNA) on pristine and highly degraded biological material. The focus is on applications to forensic casework, and a number of case examples are provided. Any laboratory working with DNA from old or “ancient” samples will benefit from this information. Curr. Protoc. Hum. Genet. 74:14.7.1‐14.7.23. © 2012 by John Wiley & Sons, Inc.

Keywords: PCR; heteroplasmy; skeletal remains; deep sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Amplification of Mitochondrial DNA
  • Basic Protocol 2: Considerations for Sequencing Mitochondrial DNA
  • Basic Protocol 3: Guidelines for the Analysis of Mitochondrial DNA Sequence Data
  • Basic Protocol 4: Second‐Generation Sequencing of Mitochondrial DNA
  • Support Protocol 1: Irradiation of Reagents and Supplies in the Ultraviolet Cross‐Linker
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Amplification of Mitochondrial DNA

  Materials
  • 10% (v/v) commercial bleach (7 mM sodium hypochlorite solution)
  • 10× PCR buffer (provided with Taq polymerase)
  • 10 mM dNTP mix ( appendix 2D)
  • 20 µg/µl DNA‐grade bovine serum albumin (BSA; appendix 2D)
  • 10 µM forward primer (see Table 14.7.1 and Table 14.7.2)
  • 10 µM reverse primer (see Table 14.7.1 and Table 14.7.2)
  • 5 U/µl AmpliTaq Gold DNA polymerase (Applied Biosystems)
  • DNA extract of any appropriate biological specimen, with accompanying reagent blank and substrate controls, if available (unit 14.3)
  • Thermal cycler (e.g., Perkin‐Elmer 9600, or 9700 operated under 9600 conditions)
  • 0.20‐ml thin‐walled PCR tubes or a 96‐well tray for PCR
  • Additional reagents and equipment for the polymerase chain reaction (Kramer and Coen, )
NOTE: Certain reagents and supplies should be UV irradiated prior to use (see protocol 5).
Table 4.7.1   MaterialsPrimer Sets Corresponding to Regions of Amplification for mtDNAmtDNA Primer Sequences

Region Primer set
CR a F15971, F15978/R599
HV1 b F15971, F15978, F15989, F16190/R16410, R16420, R16509
HV2 b F8, F15/R429, R484
PSI c F15971, F15989, F16190/R16175, R16251, R16255, R16258
PSII c F16140, F16144, F16190/ R16410, R16420
PSIII c F8, F15, F29/R270, R274, R285, R306
PSIV c F151, F153, F155/R381, R389, R429, R484
MPS1A d F15989/R16158
MPS1B d F16112/R16237
MPS2A d F16190/R16322
MPS2B d F16268/R16410
MPS3A d F34/R159
MPS3B d F109/R240
MPS4A d F151/R292
MPS4B d F220/R377
MVR1 e F16450/R16
MVR2 e F403/R569
Primer Sequence (5′ to 3′)
F15971 TTA ACT CCA CCA TTA GCA CC
F15978 CAC CAT TAG CAC CCA AAG CT
F15989 CCC AAA GCT AAG ATT CTA AT
F16112 CAC CAT GAA TAT TGT ACG GT
F16140 TAC TTG ACC ACC TGT AGT AC
F16144 TGA CCA CCT GTA GTA CAT AA
F16190 CCC CAT GCT TAC AAG CAA GT
F16268 CAC TAG GAT ACC AAC AAA CC
F16450 GCT CCG GGC CCA TAA CAC TTG
R16158 TAC TAC AGG TGG TCA AGT AT
R16175 TGG ATT GGG TTT TTA TGT A
R16237 TGT GTG ATA GTT GAG GGT TG
R16251 GGA GTT GCA GTT GAT GT
R16255 CTT TGG AGT TGC AGT TGA TG
R16258 TGG CTT TGG AGT TGC AGT TG
R16322 TGG CTT TAT GTA CTA TGT AC
R16410 GAG GAT GGT GGT CAA GGG AC
R16420 TGA TTT CAC GGA GGA TGG TG
R16509 AGG AAC CAG ATG TCG GAT AC
F8 GGT CTA TCA CCC TAT TAA CCA C
F15 CAC CCT ATT AAC CAC TCA CG
F29 CTC ACG GGA GCT CTC CAT GC
F34 GGG AGC TCT CCA TGC ATT TGG TA
F109 GCA CCC TAT GTC GCA GTA TCT GTC
F151 CTA TTA TTT ATC GCA CCT
F153 ATT ATT TAT CGC ACC TAC GT
F155 TAT TTA TCG CAC CTA CGT TC
F220 TGC TTG TAG GAC ATA ATA AT
F403 TCT TTT GGC GGT ATG CAC TTT
R16 TGA TAG ACC TGT GAT CCA TCG TGA
R159 AAA TAA TAG GAT GAG GCA GGA ATC
R240 TAT TAT TAT GTC CTA CAA GCA
R270 TGG AAA GTG GCT GTG CAG AC
R274 TGT GTG GAA AGT GGC TGT GC
R285 GTT ATG ATG TCT GTG TGG AA
R292 ATT TTT TGT TAT GAT GTC T
R306 GGG GTT TGG TGG AAA TTT TTT G
R377 GTG TTA GGG TTC TTT GTT TT
R381 GCT GGT GTT AGG GTT CTT TG
R389 CTG GTT AGG CTG GTG TTA GG
R429 CTG TTA AAA GTG CAT ACC GCC A
R484 TGA GAT TAG TAG TAT GGG AG
R569 GGT GTA TTT GGG GTT TGG TTG
R599 TTG AGG AGG TAA GCT ACA TA

 aCR, control region.
 bHV, hypervariable region.
 cPS, primer set.
 dMPS, miniprimer set.
 eMVR, minivariable region.
Table 4.7.2   MaterialsPrimer Sets Corresponding to Regions of Amplification for mtDNAmtDNA Primer Sequences

Region Primer set
CR a F15971, F15978/R599
HV1 b F15971, F15978, F15989, F16190/R16410, R16420, R16509
HV2 b F8, F15/R429, R484
PSI c F15971, F15989, F16190/R16175, R16251, R16255, R16258
PSII c F16140, F16144, F16190/ R16410, R16420
PSIII c F8, F15, F29/R270, R274, R285, R306
PSIV c F151, F153, F155/R381, R389, R429, R484
MPS1A d F15989/R16158
MPS1B d F16112/R16237
MPS2A d F16190/R16322
MPS2B d F16268/R16410
MPS3A d F34/R159
MPS3B d F109/R240
MPS4A d F151/R292
MPS4B d F220/R377
MVR1 e F16450/R16
MVR2 e F403/R569
Primer Sequence (5′ to 3′)
F15971 TTA ACT CCA CCA TTA GCA CC
F15978 CAC CAT TAG CAC CCA AAG CT
F15989 CCC AAA GCT AAG ATT CTA AT
F16112 CAC CAT GAA TAT TGT ACG GT
F16140 TAC TTG ACC ACC TGT AGT AC
F16144 TGA CCA CCT GTA GTA CAT AA
F16190 CCC CAT GCT TAC AAG CAA GT
F16268 CAC TAG GAT ACC AAC AAA CC
F16450 GCT CCG GGC CCA TAA CAC TTG
R16158 TAC TAC AGG TGG TCA AGT AT
R16175 TGG ATT GGG TTT TTA TGT A
R16237 TGT GTG ATA GTT GAG GGT TG
R16251 GGA GTT GCA GTT GAT GT
R16255 CTT TGG AGT TGC AGT TGA TG
R16258 TGG CTT TGG AGT TGC AGT TG
R16322 TGG CTT TAT GTA CTA TGT AC
R16410 GAG GAT GGT GGT CAA GGG AC
R16420 TGA TTT CAC GGA GGA TGG TG
R16509 AGG AAC CAG ATG TCG GAT AC
F8 GGT CTA TCA CCC TAT TAA CCA C
F15 CAC CCT ATT AAC CAC TCA CG
F29 CTC ACG GGA GCT CTC CAT GC
F34 GGG AGC TCT CCA TGC ATT TGG TA
F109 GCA CCC TAT GTC GCA GTA TCT GTC
F151 CTA TTA TTT ATC GCA CCT
F153 ATT ATT TAT CGC ACC TAC GT
F155 TAT TTA TCG CAC CTA CGT TC
F220 TGC TTG TAG GAC ATA ATA AT
F403 TCT TTT GGC GGT ATG CAC TTT
R16 TGA TAG ACC TGT GAT CCA TCG TGA
R159 AAA TAA TAG GAT GAG GCA GGA ATC
R240 TAT TAT TAT GTC CTA CAA GCA
R270 TGG AAA GTG GCT GTG CAG AC
R274 TGT GTG GAA AGT GGC TGT GC
R285 GTT ATG ATG TCT GTG TGG AA
R292 ATT TTT TGT TAT GAT GTC T
R306 GGG GTT TGG TGG AAA TTT TTT G
R377 GTG TTA GGG TTC TTT GTT TT
R381 GCT GGT GTT AGG GTT CTT TG
R389 CTG GTT AGG CTG GTG TTA GG
R429 CTG TTA AAA GTG CAT ACC GCC A
R484 TGA GAT TAG TAG TAT GGG AG
R569 GGT GTA TTT GGG GTT TGG TTG
R599 TTG AGG AGG TAA GCT ACA TA

 fPrimer sequences are based on the numbering system of Anderson et al. ( ). Numbering begins at the 5′ end of each primer.

Basic Protocol 2: Considerations for Sequencing Mitochondrial DNA

  Materials
  • 10× PCR buffer (provided with Taq polymerase)
  • 10 mM dNTP mix ( appendix 2D)
  • 5 U/µl AmpliTaq Gold DNA polymerase (Applied Biosystems)
  • mtDNA primer sequences for second‐generation sequencing (Table 14.7.4)
  • GS Junior Titanium emPCR Kit (LIB A) (Roche Diagnostics, cat no. 05996520001)
  • GS Junior emPCR Bead Recovery Reagents (Roche Diagnostics, cat no. 05996490001)
  • GS Junior emPCR Reagents (LIB A) (Roche Diagnostics, cat no. 05996538001)
  • GS Junior emPCR Oil & Breaking Kit (Roche Diagnostics, cat no. 05996511001)
  • GS Junior Titanium Sequencing Kit (Roche Diagnostics, cat no. 05996554001)
  • GS Junior Sequencing Reagents & Enzymes (Roche Diagnostics, cat no. 05996562001)
  • GS Junior Sequencing Buffers (Roche Diagnostics, cat no. 05996589001)
  • NextGENe software (SoftGenetics, http://www.softgenetics.com/)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
NOTE: Certain reagents and supplies should be UV irradiated prior to use (see protocol 5).
Table 4.7.4   MaterialsmtDNA Primer Sequences for Second‐Generation Sequencing

Primer Sequence (5′ to 3′)
PSI (Primer A) GCC TCC CTC GCG CCA TCA GAC G AGT GCG TCAC
F15978 CAT TAG CAC CCA AAG CT
PSI (Primer B) GCC TTG CCA GCC CGC TCA GAC GAG TGC GTCTT
R16255 TGG AGT TGC AGT TGA TG
PSII (Primer A) GCC TCC CTC GCG CCA TCA GAC G AGT GCG TAT ACT
F16139 TGA CCA CCT GT AGT AC
PSII (Primer B) GCC TTG CCA GCC CGC TCA GAC GAG TGC GTT GAT
R16420 TTC ACG GAG GAT GGT G
PSIII (Primer A) GCC TCC CTC GCG CCA TCA GAC G AGT GCG TGGT
F8 CTA TCA CCC TAT TAA CCA C
PSIII (Primer B) GCC TTG CCA GCC CGC TCA GAC GAG TGC GTGGG
R306 GTT TGG TGG AAA TTT TTT G
PSIV (Primer A) GCC TCC CTC GCG CCA TCA GAC G AGT GCG TATT
F153 ATT TAT CGC ACC TAC GT
PSIV (Primer B) GCC TTG CCA GCC CGC TCA GAC GAG TGC GTCTG
R429 TTA AAA GTG CAT ACC GCC A

 hPrimer sequences are based on the numbering system of Anderson et al. ( ). Numbering begins at the 5′ end of each PCR primer sequence. The PCR primer sequence is in BOLD font, the multiplex identifier (MID) is in ITALIC font, and the adaptor/sequencing primer sequence provided is without modification. The primers for the MID1 sequence, as listed in the 454 Life Sciences Web site (http://www.my454.com), are provided for illustration. Primers with other MID sequences will have a change to the sequence of the MID only. Primers A and B annotate which of the two 454 sequencing reactions are performed, forward or reverse.

Basic Protocol 3: Guidelines for the Analysis of Mitochondrial DNA Sequence Data

  Materials
  • Any tube constructed of polypropylene
  • UV cross‐linker (e.g., UVP Ultraviolet Cross‐linker Model CL‐1000, 254 nm)
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Figures

Videos

Literature Cited

Literature Cited
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