Table 1 Oligonucleotides used for NBN PCR amplification and sequencing.
|  | F/R1 | Oligonucleotide sequence (5'→3') | Annealing temp. (°C) | PCR lenght (bp) | MgCl2 final conc. (mM) | Comments |
|---|---|---|---|---|---|---|
Genomic sequence | ||||||
Exon 1 | F | GACGTTAAGACAAGTTGATTTGAACTTAGA* | 60 | 965/ | 0.5 | 2% DMSO added to the PCR reaction |
| Â | R | TCCGCCCATGCTAACTTCCT | Â | 1113 | Â | Â |
| Â | F2 | TTTAGTAGTGCGCAGGATGTAGAC | Â | Â | Â | Â |
Exon 2 | F | CCTTTGATAGCCTTCAGTGAGGC | 64 | 743 | 1.0 | Sequencing primer: |
| Â | R | AGCCAGAGTCATGAAGGTCTGTTC | Â | Â | Â | CCACTGGTACCACTGCCACA |
Exon 3 | F | GTCAGGAGAATCCCCACTGACTT* | 60 | 548 | 1.5 | Â |
| Â | R | GGCACAGAGTCCAATACTGTGCT | Â | Â | Â | Â |
Exon 4 | F | TGGGAAGTTACATTTCTTCGATTCC* | 58 | 728 | 1.5 | Â |
| Â | R | GCACTGTCATAACCTTCTCGGTG | Â | Â | Â | Â |
Exon 5 | F | GCAGTGACCAAAGACCGACTTCTA* | 58 | 561 | 1.5 | Â |
| Â | R | TGAGGTTACCTCAGTGCCATTTACT* | Â | Â | Â | Â |
Exon 6 | F | AAACGCGATTAGATGCTTTTTGTC* | 60 | 630 | 1.5 | Â |
| Â | R | ACCCCACTTTGGTACACAGAAC | Â | Â | Â | Â |
Exon 7 | F | CCACAGAGAGTGTAACAGTTCCAGG* | 63 | 1100 | 1.0 | 2% DMSO added to the PCR reaction |
| Â | R | TTAATTCTTGTATCGGCCGGG | Â | Â | Â | Â |
Exon 8 | F | TAACAGTGCCCCAGCGAGTAAG* | 58 | 785 | 1.5 | Â |
| Â | R | TCCTCTTACACTGTCGACCCTTAGA | Â | Â | Â | Â |
Exon 9 | F | TTAGATAAGCCCGTCATAGATGCC* | 58 | 516 | 1.5 | Â |
| Â | R | TAACTACTCGCCGCTCCTTTACA | Â | Â | Â | Â |
Exon 10 | F | GTTTGTCAGTCGTCTATAGTGGAGCA* | 58 | 763 | 1.5 | Â |
| Â | R | AATTGCGGCAAGTAAAATAACACG | Â | Â | Â | Â |
Exon 11 | F | CCCTGCCCACAACCTTACTACG* | 60 | 1500 | 1.5 | Â |
| Â | R | GACCACAGCCATGAATGAGTGG * | Â | Â | Â | Â |
Exon 12 | F | TCCTACCATCTACAGACAACCATGG | 58 | 619 | 1.5 | Â |
| Â | R | CCATGATCAATCCATTTCAAGGC * | Â | Â | Â | Â |
Exon 13 | F | GCAAACAGTGCTGAGATTTTGTGTC | 58 | 850 | 1.5 | Â |
| Â | R | CCTGAGCTAAAGAACCTCCTCAAGTAG * | Â | Â | Â | Â |
Exon 14 | F | CAACATCTCCTGCTTGGACTCTG | 60 | 638 | 1.0 | Sequencing primer: |
| Â | R | GAAGAATTTGCTTGAAGGCCACC | Â | Â | Â | GATGGGTTTAGAACAGAGTTACTGCT |
Exon 15 | F | GAACTCAGATGTGGTGACCTCCAG | 58 | 441 | 1.5 | Â |
| Â | R | CAATTTCACACAATTCGGGAACC * | Â | Â | Â | Â |
Exon 16 | F | GAGGAATGGGGATCTTTGAAGC | 58 | 457 | 1.5 | Sequencing primer: |
| Â | R | GTAACTTAAATCGCTTCTATACAC | Â | Â | Â | AAGCAACATCAAAGGGATACATGA |
cDNA | ||||||
PCR 1 | F | GTTACGCGGTTGCACGTCG | 64 | 998 | 1.5 | Exons 1 to 8 |
| Â | R | GTCATGAAAATCACCGCCAATC | Â | Â | Â | Â |
PCR 2 | F | TCTTTTTGGCTCCGGGAACG | 62 | 567 | 1.5 | Exons 7 to 10 |
| Â | R | GCTGCTGCTGAGAAGCCCTATC | Â | Â | Â | Overlap of 169 pb with PCR1 |
PCR 3 | F | CCCACTGTAAAGGAGTCCTGCA | 62 | 634 | 1.5 | Exons 10 to 12 |
| Â | R | TACTTTCTGGTACTGCTTCATCACT | Â | Â | Â | Overlap of 151 pb with PCR2 |
PCR 4 | F | CCATAGAAGATGAAGTATTGGA | 62 | 700 | 1.5 | Exons 11 to 16 |
| Â | R | GTAACTTAAATCGCTTCTATACAC | Â | Â | Â | Overlap of 165 pb with PCR3 |
Promoter cloning | ||||||
| Â | F | GACGACGCTAGCGACGTTAAGACAAGTTGATTTGAACTTAGA | 62 | 515 | 1.0 | 2% DMSO added to the PCR reaction Added restriction sites are underlined |
| Â | R | GACGACAAGCTTATCGGTCCGGCTCCTCAGGGCTG | Â | Â | Â | Â |