Table 1

Oligonucleotides (A) and conditions (B) for UPQFM-PCR

PCR reactionRegion amplifiedSense primerAntisense primerAmount of primer added to volume of 25 μl (pmol)Product length
Each primer used in PCR 1 reaction begins with 22 bp P2 universal sequence.
A fluorescent label (6-Fam) is attached to the 5` end of the antisense P2 universal primer only.
(A) Oligonucleotides for UPQFM-PCR
PCR 1Exon 1E1Fn:P2-UnivF-tacggccctgaagaagacg5321 bp
Exon 2E2Fn:P2-UnivF-agacgaggtttcaccacgtt2396 bp
Exon 3E3Fn:P2-UnivF-tactgagaccctagtctgtcactg1,5364 bp
β-globinBgFn:P2-UnivF-cacaccctagggttggccaa2304 bp
PCR 2All above fragmentsP2 Univ F:tcc gtc tta gct gag tgg cgt a4
P2 Univ R:6-Fam-agg cag aat cga ctc acc gct a4
PCR reactionInitial denaturation at 94°CEach cycle ofNo of cyclesFinal elongation at 72°CTemplate
Denaturation at 95°CAnnealing at 60°CElongation at 72°C
(B) Conditions for UPQFM-PCR
PCR-110 minutes1 minute2 minutes1 minute9 cycles5 minutes100 ng genomic DNA
PCR-24 minutes1 minute2 minutes1 minute20 cycles5 minutes2 μl PCR 1 product