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

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 College of Medicine Human Identification Research Center, Yonsei University 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 1

AFDIL mini-primer set 16569 / 1 16569 / 1 16569 / 1 16569 / 1 16569 / 1 HV1 (342 bp) HV1 (342 bp) HV1 (342 bp) HV1 (342 bp) HV2 (268 bp) HV2 (268 bp) HV2 (268 bp) HV2 (268 bp) 16024 16024 16024 16024 16365 16365 16365 16365 73 73 73 73 340 340 340 340 F15989 F15989 F15989 F15989 F034 F034 F034 F034 Mps1a (170 bp) Mps1a (170 bp) Mps1a (170 bp) Mps1a (170 bp) Mps3a (126 bp) Mps3a (126 bp) Mps3a (126 bp) Mps3a (126 bp) R16158 R16158 R16158 R16158 R159 R159 R159 R159 F16112 F16112 F16112 F16112 F16112 F109 F109 F109 F109 Mps1b (140 bp) Mps1b (140 bp) Mps1b (140 bp) Mps1b (140 bp) Mps1b (140 bp) Mps3b (132 bp) Mps3b (132 bp) Mps3b (132 bp) Mps3b (132 bp) 16189 C-stretch 16189 C-stretch R16251 R16251 R16251 R16251 R16251 R240 R240 R240 R240 F16190 F16190 F16190 F16190 F16190 F151 F151 F151 F151 Mps2a (133 bp) Mps2a (133 bp) Mps2a (133 bp) Mps2a (133 bp) Mps2a (133 bp) Mps4a (142 bp) Mps4a (142 bp) Mps4a (142 bp) Mps4a (142 bp) R16322 R16322 R16322 R16322 R16322 R292 R292 R292 R292 F16268 F16268 F16268 F16268 F16268 F220 F220 F220 F220 Mps2b (143 bp) Mps2b (143 bp) Mps2b (143 bp) Mps2b (143 bp) Mps2b (143 bp) Mps4b (170 bp) Mps4b (170 bp) Mps4b (170 bp) Mps4b (170 bp) C-stretch C-stretch R16410 R16410 R16410 R16410 R16410 R389 R389 R389 R389 Gabriel MN et al. (2001) J Forensic Sci. 46:247-253. � 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. 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. 2

The modified mini-primer sequence Control region Amplicon Primer sequence (5 ′ → 3 ′ ) Amplicon size F15989 CCC AAA GCT AAG ATT CTA AT M11 165bp R16153 CAG GTG GTC AAG TAT TTA TGG F16097 TAC ATT ACT GCC AGC CAC CA M12 137bp R16233 TGA TAG TTG AAG GTT GAT TGC TGT HV1 F16159 CAT AAA AAC CCA ATC CAC AT M13 146bp R16304 ACT GTT AAG GGT GGG TAG GT F16247 ACT CCA AAG CCA CCC CTC A M14 164bp R16410 GAG GAT GGT GGT CAA GGG AC F015 CAC CCT ATT AAC CAC TCA CG M21 173bp R187 CGC CTG TAA TAT TGA ACG TA F120 CGC AGT ATC TGT CTT TGA TTC C HV2 M22 166bp R285 GTT ATG ATG TCT GTG TGG AA F220 TGC TTG TAG GAC ATA ATA AT M23 170bp R389 CTG GTT AGG CTG GTG TTA GG PCR amplification efficiency test � DNA samples • 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 • Total 25.0 μ l PCR reaction : 2 μ l of DNA, 2.5 μ l of STR buffer, 2.5 U AmpliTaq Gold polymerase and primer � Thermal Cycling 95 ℃ for 11 min 95 ℃ for 20 sec 50 ℃ for 20 sec X 42 cycles (bone) 72 ℃ for 30 sec Blood sample : annealing Tm - 56 ℃ , 35 cycles 72 ℃ for 7 min 3

Comparison of the PCR amplification efficiency A. AFDIL mini-primer A. AFDIL mini-primer HV1 HV1 HV1 HV2 HV2 HV2 M Mps1a Mps1b Mps2a Mps2b Mps3a M Mps1a Mps1b Mps2a Mps2b Mps3a M Mps1a Mps1b Mps2a Mps2b Mps3a Mps3b Mps4a Mps4b Mps3b Mps4a Mps4b Mps3b Mps4a Mps4b B. modified mini-primer B. modified mini-primer HV1 HV1 HV1 HV2 HV2 HV2 M M11 M12 M13 M14 M M11 M12 M13 M14 M M11 M12 M13 M14 M21 M22 M23 M21 M22 M23 M21 M22 M23 PCR amplification efficiency on mutation samples 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% 16140 mutation 16140 mutation 16140 mutation 16140 mutation 16209 mutation 16209 mutation 16209 mutation 16209 mutation 16304 mutation 16304 mutation 16304 mutation M M M Mps1a Mps1a Mps1a Mps1a M11 M11 M11 M11 Mps2a Mps2a Mps2a Mps2a M13 M13 M13 M13 Mps2a Mps2a Mps2a M13 M13 M13 4

Comparison of the sequence alignment with the 16189 mutation sample A. AFDIL mini-primer A. AFDIL mini-primer sequencing gap B. Modified mini-primer B. Modified mini-primer The modified mini-primer set 16569 / 1 16569 / 1 16569 / 1 16569 / 1 HV1 (342 bp) HV1 (342 bp) HV1 (342 bp) HV2 (268 bp) HV2 (268 bp) HV2 (268 bp) 16024 16024 16024 16365 16365 16365 73 73 73 340 340 340 F15989 F15989 F15989 F15989 F15989 F015 F015 F015 F015 F015 M11 (165 bp) M11 (165 bp) M11 (165 bp) M11 (165 bp) M11 (165 bp) M21 (173 bp) M21 (173 bp) M21 (173 bp) M21 (173 bp) M21 (173 bp) R16153 R16153 R16153 R16153 R16153 R187 R187 R187 R187 R187 F16097 F16097 F16097 F16097 F16097 F16097 F120 F120 F120 F120 F120 M12 (137 bp) M12 (137 bp) M12 (137 bp) M12 (137 bp) M12 (137 bp) M12 (137 bp) M22 (166 bp) M22 (166 bp) M22 (166 bp) M22 (166 bp) M22 (166 bp) R16233 R16233 R16233 R16233 R16233 R16233 R285 R285 R285 R285 R285 F16159 F16159 F16159 F16159 F16159 F16159 F220 F220 F220 F220 F220 M13 (146 bp) M13 (146 bp) M13 (146 bp) M13 (146 bp) M13 (146 bp) M13 (146 bp) M23 (170 bp) M23 (170 bp) M23 (170 bp) M23 (170 bp) M23 (170 bp) 16189 C-stretch 16189 C-stretch 16189 C-stretch C-stretch C-stretch C-stretch R16304 R16304 R16304 R16304 R16304 R16304 R389 R389 R389 R389 R389 F16247 F16247 F16247 F16247 F16247 F16247 M14 (164 bp) M14 (164 bp) M14 (164 bp) M14 (164 bp) M14 (164 bp) M14 (164 bp) R16410 R16410 R16410 R16410 R16410 R16410 � 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. 5

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. 6

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). 7