A modified mini-primer set for the mtDNA control region sequence - - PDF document

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a modified mini primer set for the mtdna control region
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A modified mini-primer set for the mtDNA control region sequence - - PDF document

Apr 27, 2023 217 likes •301 views

A modified mini-primer set for the mtDNA control region sequence analysis from highly degraded forensic remains Na Young Kim, Hwan Young Lee, Myung Jin Park, Woo Ick Yang, Kyoung-Jin Shin Department of Forensic Medicine, Yonsei University

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A modified mini-primer set for the mtDNA control region sequence analysis from highly degraded forensic remains

Department of Forensic Medicine, Yonsei University College of Medicine Human Identification Research Center, Yonsei University

Na Young Kim, Hwan Young Lee, Myung Jin Park, Woo Ick Yang, Kyoung-Jin Shin

Mitochondrial DNA

General properties

  • Maternal inheritance
  • High copy number per cell

→ Powerful tool for forensic identity testing of highly degraded skeletal remains

Control region : major target for forensic field

  • Hypervariable regions (HV1, HV2 and HV3)
  • Length heteroplasmy and point heteroplasmy
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AFDIL mini-primer set

The different annealing temperature and times for primer pairs make mtDNA analysis labor-intensive. A sequence gap on samples with the HV1 length heteroplasmy. AFDIL mini-primer produces reduced or inhibited amplification yield by some frequent mutation.

Mps3a (126 bp) F034 R159 F109 R240 Mps3b (132 bp) F151 R292 Mps4a (142 bp) F220 R389 Mps4b (170 bp) F16112 R16251 Mps1b (140 bp) F16268 R16410 Mps2b (143 bp) F15989 R16158 Mps1a (170 bp) F16190 R16322 Mps2a (133 bp)

HV1 (342 bp) HV2 (268 bp) 16024 16365 73 340 16569 / 1 16189 C-stretch C-stretch

Mps3a (126 bp) F034 R159 F109 R240 Mps3b (132 bp) F151 R292 Mps4a (142 bp) F220 R389 Mps4b (170 bp) F16112 R16251 Mps1b (140 bp) F16268 R16410 Mps2b (143 bp) F15989 R16158 Mps1a (170 bp) F16190 R16322 Mps2a (133 bp)

HV1 (342 bp) HV2 (268 bp) 16024 16365 73 340 16569 / 1

Mps3a (126 bp) F034 R159 F109 R240 Mps3b (132 bp) F151 R292 Mps4a (142 bp) F220 R389 Mps4b (170 bp) F16112 R16251 Mps1b (140 bp) F16268 R16410 Mps2b (143 bp) F15989 R16158 Mps1a (170 bp) F16190 R16322 Mps2a (133 bp) Mps3a (126 bp) F034 R159 F109 R240 Mps3b (132 bp) F151 R292 Mps4a (142 bp) F220 R389 Mps4b (170 bp) F16112 R16251 Mps1b (140 bp) F16112 R16251 Mps1b (140 bp) F16268 R16410 Mps2b (143 bp) F16268 R16410 Mps2b (143 bp) F15989 R16158 Mps1a (170 bp) F16190 R16322 Mps2a (133 bp) F16190 R16322 Mps2a (133 bp)

HV1 (342 bp) HV2 (268 bp) 16024 16365 73 340 16569 / 1 HV1 (342 bp) HV2 (268 bp) 16024 16365 73 340 16569 / 1 16569 / 1 16189 C-stretch C-stretch

Gabriel MN et al. (2001) J Forensic Sci. 46:247-253.

Primer design of modified mini-primer set

Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)

  • Size : 18 - 27 nucleotide (optimum : 20 nucleotide)
  • Temperature : 55℃ - 63℃ (optimum : 60℃)
  • GC content : 20% - 80%

→ Screening PCR The final set of primers with high amplification efficiency, relatively short primer length, and high Tm.

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The modified mini-primer sequence

Control region Amplicon Primer sequence (5′ → 3′) Amplicon size F15989 CCC AAA GCT AAG ATT CTA AT M11 R16153 CAG GTG GTC AAG TAT TTA TGG 165bp F16097 TAC ATT ACT GCC AGC CAC CA M12 R16233 TGA TAG TTG AAG GTT GAT TGC TGT 137bp F16159 CAT AAA AAC CCA ATC CAC AT M13 R16304 ACT GTT AAG GGT GGG TAG GT 146bp F16247 ACT CCA AAG CCA CCC CTC A HV1 M14 R16410 GAG GAT GGT GGT CAA GGG AC 164bp F015 CAC CCT ATT AAC CAC TCA CG M21 R187 CGC CTG TAA TAT TGA ACG TA 173bp F120 CGC AGT ATC TGT CTT TGA TTC C M22 R285 GTT ATG ATG TCT GTG TGG AA 166bp F220 TGC TTG TAG GAC ATA ATA AT HV2 M23 R389 CTG GTT AGG CTG GTG TTA GG 170bp

PCR amplification efficiency test

DNA samples

  • Total 25.0 μl PCR reaction : 2 μl of DNA, 2.5 μl of STR buffer, 2.5 U AmpliTaq Gold

polymerase and primer

  • mtDNA haplogroup D4 (16223-16362-73-263-315.1C)
  • HV1 length heteroplasmy
  • Mutations are located at AFDIL’s mini-primer annealing site

→ Long bone and molar teeth samples from 55 year-old skeletal remains DNA from blood samples

PCR amplification Thermal Cycling

95℃ for 11 min 95℃ for 20 sec 50℃ for 20 sec 72℃ for 30 sec 72℃ for 7 min X 42 cycles (bone) Blood sample : annealing Tm - 56℃, 35 cycles

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Comparison of the PCR amplification efficiency

M Mps1a Mps1b Mps2a Mps2b Mps3a Mps3b Mps4a Mps4b HV1 HV2 M M11 M12 M13 M14 M21 M22 M23 HV1 HV2

  • A. AFDIL mini-primer
  • B. modified mini-primer

M Mps1a Mps1b Mps2a Mps2b Mps3a Mps3b Mps4a Mps4b HV1 HV2 M Mps1a Mps1b Mps2a Mps2b Mps3a Mps3b Mps4a Mps4b HV1 HV2 M M11 M12 M13 M14 M21 M22 M23 HV1 HV2 M M11 M12 M13 M14 M21 M22 M23 HV1 HV2

  • A. AFDIL mini-primer
  • B. modified mini-primer

PCR amplification efficiency on mutation samples

Mps1a M11 M Mps2a M13 Mps2a M13 16140 mutation 16209 mutation 16304 mutation Mps1a M11 M Mps2a M13 Mps2a M13 16140 mutation 16209 mutation 16304 mutation Mps1a M11 Mps1a M11 M Mps2a M13 Mps2a M13 Mps2a M13 16140 mutation 16140 mutation 16209 mutation 16209 mutation 16304 mutation

Mutation frequencies

Population n.p. 16140 n.p. 16209 n.p. 16304 Caucasians 0.2% 1.3% 8.6% Asians 5.1% 3.2% 14.7% African Americans 0.3% 6.3% 0.9%

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Comparison of the sequence alignment with the 16189 mutation sample

  • A. AFDIL mini-primer
  • B. Modified mini-primer
  • A. AFDIL mini-primer
  • B. Modified mini-primer

sequencing gap

The modified mini-primer set

The modified mini-primer set is composed of four and three PCR amplicons. The forward primer of M13 was designed to be avoid the possible gap on samples with the HV1 length heteroplasmy. Using the FBI mtDNA population database, the first to the third nucleotide from the 3’ end of each primer were located at the nucleotide position with a low mutation frequency in the Caucasians, Asians, and Africans.

HV1 (342 bp) HV2 (268 bp) 16024 16365 73 340 16569 / 1

F015 R187 M21 (173 bp) F120 R285 M22 (166 bp) F220 R389 M23 (170 bp) F15989 R16153 M11 (165 bp) F16097 R16233 M12 (137 bp) F16159 R16304 M13 (146 bp) F16247 R16410 M14 (164 bp)

16189 C-stretch C-stretch HV1 (342 bp) HV2 (268 bp) 16024 16365 73 340 16569 / 1 HV1 (342 bp) HV2 (268 bp) 16024 16365 73 340 16569 / 1 16569 / 1

F015 R187 M21 (173 bp) F120 R285 M22 (166 bp) F220 R389 M23 (170 bp) F15989 R16153 M11 (165 bp) F16097 R16233 M12 (137 bp) F16159 R16304 M13 (146 bp) F16247 R16410 M14 (164 bp)

16189 C-stretch C-stretch

F015 R187 M21 (173 bp) F120 R285 M22 (166 bp) F220 R389 M23 (170 bp) F15989 R16153 M11 (165 bp) F16097 R16233 M12 (137 bp) F16159 R16304 M13 (146 bp) F16247 R16410 M14 (164 bp) F015 R187 M21 (173 bp) F120 R285 M22 (166 bp) F220 R389 M23 (170 bp) F15989 R16153 M11 (165 bp) F16097 R16233 M12 (137 bp) F16159 R16304 M13 (146 bp) F16247 R16410 M14 (164 bp) F015 R187 M21 (173 bp) F120 R285 M22 (166 bp) F220 R389 M23 (170 bp) F15989 R16153 M11 (165 bp) F16097 R16233 M12 (137 bp) F16097 R16233 M12 (137 bp) F16159 R16304 M13 (146 bp) F16159 R16304 M13 (146 bp) F16247 R16410 M14 (164 bp) F16247 R16410 M14 (164 bp)

16189 C-stretch C-stretch

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Alternative primers

In Africans

n.p. 16265 mutation frequency : 6.5%

M14 - F16247 → F16255 (5'-GCC ACC CCT CAC CCA CTA G) M13 - R16304 → R16322 (5'-TGG CTT TAT GTA CTA TGT AC)

In Caucasians

n.p. 239 mutation frequency : 1.9%

M23 – F220 → F220* (5'-TGC TTG TAG GAC ATA ATA ATA ACA A)

Conclusion

We developed the modified mini-primer set which makes up for the weak points of AFDIL mini-primer set.

  • HV1 length heteroplasmy
  • Nucleotide variability
  • PCR amplification condition

The modified mini-primer set will be a useful tool for mtDNA control region sequence analysis from highly degraded forensic samples.

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Acknowledgements

This work was supported by grant number M10640010002- 16N4001-00210 from the Korean Ministry of Science and Technology (MOST) and the Korean Science and Engineering Foundation (KOSEF), and grants from MAKRI (Ministry of Naional Defense Agency for Killed In Action Recovery and Identification).