Fig 1.
PCR-APLP method for SNP analysis.
(A) and (B) Desired extension by specific hybridization of the competitive primer to the SNP site. (C) Unexpected extension by non-specific hybridization of the competitive primer to the SNP site and its electrophoretic banding pattern.
Table 1.
Primers used to assess the binding properties of inosine and the electrophoretic mobility of the resulting amplicon.
Table 2.
Primers used for evaluating the effectiveness of inosine in improving competitiveness.
Table 3.
Tm values of competing primers.
Fig 2.
Sequence electropherogram (upper panel) and electrophoretic pattern (lower panel) of the amplicon generated using the inosine flap primer.
The sequence data is of the amplicon obtained by the Inosine H1047 primer with five inosines added to the 5′-terminus (Table 1). LM indicates 10 bp ladder marker.
Fig 3.
Thermodynamics of allele-specific primers.
PCR reactions were carried by using N and F primer sets at various annealing temperatures from 60°C to 70°C. PCR amplification conditions are described in Materials and Methods. Upper panel shows electrophoretogram of the amplicon generated by the inosine flap primer and competitive allele-specific primer. Lower panel shows electrophoretogram of the amplicon generated using the primer with a flap of ordinary non-complemental bases and a competitive allele-specific primer. Sample 1 indicates human mtDNA haplogroup non-N and non-F, and sample 2 indicates human mtDNA haplogroup F. Haplogroup F is a branch of macrohaplogroup N; therefore, this mtDNA is also assigned to haplogroup N. LM indicates 10 bp ladder marker.
Fig 4.
Competitiveness and sensitivity of allele-specific primers.
MtDNA templates contained mixtures of haplogroups M7a (non-N and non-F; Sample 1) or F (sample 2) mtDNA. Templates (1.0 × 10−9–0.1 × 10−19 g) were amplified using N and F primer sets as shown in Materials and Methods. Upper panel shows electrophoretogram of the amplicons generated using the inosine flap primer and the competitive allele-specific primer. Lower panel shows electrophoretogram of the amplicons generated using a primer with a flap of ordinary non-complementary bases and a competitive allele-specific primer. PCR amplification conditions are described in Materials and Methods. NC indicates negative PCR control. LM indicates 10 bp ladder marker.
Fig 5.
Comparison between PCR-APLP SNP analyses using the inosine flap primer and primer with a flap of ordinary non-complementary bases.
Yellow, blue, red, and white open boxes indicate human mtDNA haplogroups N, R, F, and N9, respectively. LM indicates 10 bp ladder marker. Lanes 1 and 6: haplogroup D4a mtDNA; lanes 2 and 7: haplogroup B4a mtDNA; lanes 3 and 8: haplogroup F1b mtDNA; lanes 4 and 9, haplogroup N9b mtDNA; and lanes 5 and 10, haplogroup A mtDNA.
Fig 6.
Melting curves of primer-probe pairs of N, R, N9, F and A sets, shown in Table 3.
Melting curves of primers are shown as coloured lines as follows: black ‘Primer with inosine (I) and Prb-α-1st’, red ‘Primer with I and Prb-β-1st’, green ‘Primer with I and Prb-α-IF-2nd, purple ‘Primer with a flap of ordinary non-complementary bases and Prb-α-1st’, blue ‘Primer with a flap of ordinary non-complementary bases and Prb-β-1st’, orange ‘Primer with a flap of ordinary non-complementary bases and Prb-α-NF-2nd’, pink ‘Primer without a flap and Prb-α-1st and 2nd’, and yellow-green ‘Primer without a flap and Prb-β-1st and 2nd’. Each Tm value shown in Table 3 was obtained from the melting curves by differentiation. Values are means for triplicate assays.