Table 1.
Genomic and complementary DNA primer pairs, product length and annealing temperatures used for the amplification of exons in the PFKM gene.
| Exon amplification | Forward (5′-3′) primer | Reverse (5′-3′) primer | Annealing temperature | Product length | |
|---|---|---|---|---|---|
| cDNA Primers | A (Exons 1-4) | GTGTGTGATTGAGTCTCGCGGGT | ACCAGAGGTTAACACGGCGATGG | 56 °C | 250 bpa |
| G (Exons 11-17) | GTGGCTGAGGGTGCGATTGACA | GTTTGGAACCGCCTTGGCCAGT | 60 °C | 622 bp | |
| H (Exons 17-21) | ATCGAGGAGGCTGGCTGGAG | CAGGCCCCTCTTCACAGTTGTTTT | 60 °C | 531 bp | |
| I (Exons 20-25) | CGGATGCTGCCTACATTTT | TTTTCACAGTGACCAGTTGGCATTTA | 56 °C | 977 bp | |
| gDNA Primers | B (Exon 4) | GAGCCTGACTGAGATGGCTCTTGC | ACCCAGTCCCTCCTCAAGCAGA | 56 °C | 228 bp |
| C (Exon 5) | TCCCAGATTGCCCTGTGAGGAA | CAGCATGTAACTGGGAGGTGAGGA | 56 °C | 400 bp | |
| D (Exon 6) | TGTCATGGGCATCCAGGACTCA | AGAGCAATCGTGGCACCTCTCT | 56 °C | 372 bp | |
| E (Exons 7-8) | AGGAGTGACCCTGTGGTAGCTCAG | GGTGAGAAGACGCAAGCCTACAGC | 61 °C | 969 bp | |
| F (Exons 9-12) | TCTCAGGGAGCCTGCTGGAAGATC | GTGCAGTCGTGCAAGTTGTGGGTC | 61 °C | 1287 bp |
a
Primer pair A works only with testicular cDNA because the forward primer lies within exon 1 which is not present in blood cDNA due to tissue-specific differential splicing. Also the product length 250 bp is based on the testicular cDNA which does not include the 14 bp of exon 3 because it is spliced out in the testicular transcript.