Table 3.
Primer analysis
| Primer | Sequence | Degeneracy | Rearranged V | Germline V |
|---|---|---|---|---|
| 5′ set | ||||
| VR35-1.4 | Gccacrtattactgtg | 2 | 123 (72, 51) | 3 |
| VR35-2.4 | Gccatntattactgtg | 4 | 434 (107, 30, 258, 39) | 3 |
| VR35-3.4 | Gccgthtattactgtg | 3 | 577 (356, 148, 73) | 1 |
| VR35-4.4 | Gccttgtattactgtg | 1 | 66 | 2 |
| VR35-5.4 | Gctgthtattactgtg | 3 | 391 (120, 162, 109) | 0 |
| VR35-6.4 | Gcygtgtattactgtg | 2 | 2118 (974, 1144) | 35 |
| VR35-7.4 | GCYGTVTATTATTGTG | 6 | 303 (33, 36, 95, 23, 49, 67) | 0 |
| VR35-8.4 | GCYGTNTATTTCTGTG | 8 | 170 (17, 23, 37, 17, 8, 13, 47, 9) | 0 |
| Total | 4182/5646 74% | 44/49 90% | ||
| 3′ set | ||||
| JH1.4-3′ | CCAGGGTGCCCTGGCCCCA | 1 | 170 | JH4, JH5 |
| JH2.4-3′ | CCAGGGTGCCACGGCCCCA | 1 | 94 | JH6 |
| JH3.4-3′ | CCATTGTCCCTTGGCCCCA | 1 | 487 | JH3 |
| JH4.4-3′ | CCAGGGTTCCCTGGCCCCA | 1 | 1958 | JH1 |
| JH6.4-3′ | CCGTGGTCCCTTGGCCCCA | 1 | 802 | JH2 |
| Total | 3511/5646 62% | 6/6 100% | ||
Sequences of those portions of the 5′ and 3′ primers corresponding to the VH or JH genes suitable for the amplification of HCDR3s are shown. These were analyzed by using only the portion of the primer which recognizes the V or J gene and searching against the database of 5646 rearranged V genes or the 49 germline V genes or 6 JH genes. These analyses are stringent (100%), so it is likely that in real experimental situations, more sequences are likely to be amplified, as in each case the 3′ primer sequences are extremely well conserved. Under ‘Rearranged V’, the total numbers of the 5646 rearranged VH genes downloaded from IMGT with absolute homology to each of the primers are given. In brackets are given the number of VH genes recognized by each of the individual primers making up the degenerate pool. Under ‘Germline V’, the number of germline VH genes with 100% homology to the primers is given.