Table 2.
PCR Primers
| Assay | Primer names | Sequence or reference | Notes |
|---|---|---|---|
| Microsatellite instability | |||
| mBat-37 | Bacher et al.53 | ||
| A27 | Kabbarah et al.54 | ||
| (GT)3 tract of Tgfbr2 | Lowsky et al.55 | ||
| (AAAAG)2 tract of Tgfbr2 | TGFBR2-Fwd | 5′-GACCCCAAGCTCACCTACCAC-3′ | 35 cycles of 95°C for 1 minute, 62°C for 30 seconds, 72°C for 30 seconds |
| TGFBR2-Rev2 | 5′-TACCCGGGATAACTACTCACCTTC-3′ | ||
| D7Mit123 | UniSTS Accession No. 116680 | 50 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds | |
| D7Mit91 | UniSTS Accession No. 131298 | 30 cycles of 94°C for 60 seconds, 56°C for 60 seconds, and 72°C for 30 seconds | |
| U12235 | A hemi-nested PCR was performed using primers P1 and P256 for 25 rounds of 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 2 minutes, followed by 35 rounds with primers P1 and P3 of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds | ||
| Quantitative RT-PCR | |||
| Mouse AID | QRT-PCR-mAID.up2 | 5′-CTGGGAAGGGCTACATGAAA-3′ | 95°C for 15 minutes followed by 40 cycles of 94°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds |
| QRT-PCR-mAID.down2 | 5′-GAAATGCATCTCGCAAGTCA-3′ | ||
| Mouse GAPDH | QRT-PCR-mGAPDH.fwd | 5′-GGCATTGCTCTCAATGACAA-3′ | |
| QRT-PCR-mGAPDH.rev | 5′-CCCTGTTGCTGTAGCCGTAT-3′ | ||
| Somatic hypermutation | |||
| JH2-JH4 intron | JH2-JH4.fwd | 5′-GGCACCACTCTCACAGTCTCCTCAGG-3′ | 95°C for 5 minutes followed by 35 cycles of 95°C for 30 seconds, 60°C for 1 minute, 72°C for 1 minute |
| JH2-JH4.Adown | 5′-CCTGACCCAGACCCATGTCTCAACTTT-3′ | ||
| V(D)J recombination | |||
| Heavy chain locus | As per Ehlich et al.51 | Hot-start PCR | |
| Light chain V-Jκ | As per Yamagami et al.52 | ||