Table 1.
Primers and conditions for the tetra-primer amplification refractory mutation system–polymerase chain reaction (T-ARMS–PCR) cytotoxic T lymphocyte antigen-4 (CTLA-4) polymorphisms.
| SNP | Name1 | Primer sequences2 | Conc3 (µM) | Amplicon size (bp)4 | Ta5 |
|---|---|---|---|---|---|
| −318 C/T | 318Fo | 5′-CAATGAAATGAATTGGACTGGATG-3′ | 0·5 | K 296 | 58°C |
| 318Ro | 5′-TGCACACACAGAAGGCTCTTGAATA-3′ | 0·5 | C 201 | ||
| 318Fi(C) | 5′-CTCCACTTAGTTATCCAGATCTTC-3′ | 0·8 | T 141 | ||
| 318Ri(T) | 5′-ACTGAAGCTTCATGTTCACTCTA-3′ | 1·0 | |||
| +49 A/G | 49Fo | 5′-GTGGGTTCAAACACATTTCAAAGCTTCAGG-3′ | 0·25 | K 229 | 62°C |
| 49Ro | 5′-TCCATCTTCATGCTCCAAAAGTCTCACTC-3′ | 0·5 | A 162 | ||
| 49Fi(G) | 5′-GCACAAGGCTCAGCTGAACCTGGATG-3′ | 2·0 | G 120 | ||
| 49Ri(A) | 5′-ACAGGAGAGTGCAGGGCCAGGTCCTAGT-3′ | 1·0 |
F: forward; R: reverse. o: outer (common). i: inner (allele-specific). The nucleotide specificity is indicated in parentheses.
GenBank sequence ID number M74363. Deliberate mismatches (shown in italics) have been introduced to increase the specificity of the allele-specific primers.
The concentration (Conc) of each primer has been determined experimentally to increase specificity and to produce similar amplification of all amplicons.
K size of the control amplicon (shown in italics).
Ta: annealing temperature. Cycling conditions are 10 min at 95°C followed by 35 cycles of 94°C for 30 s, Ta (see above) for 30 s, 72°C for 30 s and a final cycle 72°C for 7 min.