Table 2

Primer combination and optimal PCR conditions used to genotype 142C→G (m1), 355G→T (m2), 4326C→G (m3), 4390A→G (m4), and 4390C→G (m5), as well as linkage disequilibrium analysis (see Materials and Methods)

SNPPCR TypePrimers UsedAnnealing TempMgCl2No. of cyclesFragment length
°C mM bp
m1PCR 1ex2F + ex2R601.335914
PCR 2Fm1wt/Fm1mt + 1/2mutR541.315416
m2PCR 1ex2F + ex2R601.335914
PCR 2Fm2wt/Fm2mt + 1/2mutR541.315203
m3PCR 1ex3F + ex3R601.335270
PCR 2ex3F +m3Rwt/m3Rmt541.320153
m4PCR 1ex3F +ex3R601.335270
PCR 2ex3F +Rm4wt/Rm4mt541.320217
m5PCR 1ex3F +ex3R601.335270
PCR 2ex3F +Rm5wt/Rm5mt541.320186
m1 vs. m2PCR 1ex2F +ex2R601.335914
PCR 2Fm1wt +Rm2wt560.820242
Fm1wt + Rm2mt
Fm1mt + Rm2wt
Fm1mt + Rm2mt
m3 vs. m4PCR 1ex3F + ex3R601.335270
PCR 2Fm3wt + Rm4wt540.92093
Fm3wt + Rm4mt
Fm3mt + Rm4wt
Fm3mt + Rm4mt
m3 vs. m5PCR 1ex3F + ex3R601.335270
PCR 2Fm3wt/Fm3mt + ex3R followed by BstNI RFLP541.320146
m1 vs. m3, m4 or m5PCR 1m1Fwt/m1Fmt + 1B13R (allele-specific long PCR) followed by genotyping for m3, m4 or m5 as described above in PCR II680.8–1.0304695