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)

SNP	PCR Type	Primers Used	Annealing Temp	MgCl₂	No. of cycles	Fragment length
			°C	mM		bp
m1	PCR 1	ex2F ⁺ ex2R	60	1.3	35	914
	PCR 2	Fm1wt/Fm1mt ⁺ 1/2mutR	54	1.3	15	416
m2	PCR 1	ex2F ⁺ ex2R	60	1.3	35	914
	PCR 2	Fm2wt/Fm2mt ⁺ 1/2mutR	54	1.3	15	203
m3	PCR 1	ex3F ⁺ ex3R	60	1.3	35	270
	PCR 2	ex3F ⁺m3Rwt/m3Rmt	54	1.3	20	153
m4	PCR 1	ex3F ⁺ex3R	60	1.3	35	270
	PCR 2	ex3F ⁺Rm4wt/Rm4mt	54	1.3	20	217
m5	PCR 1	ex3F ⁺ex3R	60	1.3	35	270
	PCR 2	ex3F ⁺Rm5wt/Rm5mt	54	1.3	20	186
m1 vs. m2	PCR 1	ex2F ⁺ex2R	60	1.3	35	914
	PCR 2	Fm1wt ⁺Rm2wt	56	0.8	20	242
		Fm1wt ⁺ Rm2mt
		Fm1mt ⁺ Rm2wt
		Fm1mt ⁺ Rm2mt
m3 vs. m4	PCR 1	ex3F ⁺ ex3R	60	1.3	35	270
	PCR 2	Fm3wt ⁺ Rm4wt	54	0.9	20	93
		Fm3wt ⁺ Rm4mt
		Fm3mt ⁺ Rm4wt
		Fm3mt ⁺ Rm4mt
m3 vs. m5	PCR 1	ex3F ⁺ ex3R	60	1.3	35	270
	PCR 2	Fm3wt/Fm3mt ⁺ ex3R followed by BstNI RFLP	54	1.3	20	146
m1 vs. m3, m4 or m5	PCR 1	m1Fwt/m1Fmt ⁺ 1B13R (allele-specific long PCR) followed by genotyping for m3, m4 or m5 as described above in PCR II	68	0.8–1.0	30	4695