Experimental conditions for RAS mutation analysis, screening by denaturing high-performance liquid chromatography (DHPLC), and restriction fragment length polymorphism (RFLP) analysis quantification of mutant allele percentage by PCR with an end-labeled radioactive primer
Method type/assay . | Primer sequence . | Annealing temperature, °C . | No. PCR cycles . | Amplicon size, bp . | RFLP enzyme . | RFLP digest product sizes, bp . |
|---|---|---|---|---|---|---|
| Mutation screening | ||||||
| DHPLC | ||||||
| K61F* | 5′-tgt gtt tct ccc ttc tca gga ttc-3′ | 60 | 35 | 158 | NA | NA |
| K61R | 5′-tgg caa ata cac aaa gaa agc c-3′ | 60 | 35 | 158 | NA | NA |
| K61F* | 5′-gat tcc tac cgg aag cag gt-3′ | 60 | 35 | 151 | NA | NA |
| K61R | 5′-tgg tgt tgt tga tgg caa ac-3′ | 60 | 35 | 151 | NA | NA |
| RFLP and sequencing | ||||||
| K12F (MM) | 5′-gac tga ata taa act tgt ggt agt tgg acc t-3′ | 56 | 35 | 121 | BstN1 (7.5 units) | Wt = 91 |
| K12R | 5′-aac aag att tac ctc tat tgt tgg atc a-3′ | 56 | 35 | 121 | (7.5 units) | Mut = 121 |
| H12F | 5′-agg aga ccc tgt agg agga-3′ | 60 | 35 | 170 | Msp1 (10 units) | Wt = 23, 56, 91 |
| H12R | 5′-gcg cta ggc tca cct cta t-3′ | 60 | 35 | 170 | (10 units) | Mut = 23, 147 |
| Mutant allele quantification: PCR with a end-labeled radioactive primer | ||||||
| N12F (MM) | 5′-act gag tac aaa ctg gtg gtg gtt gga cca-3′ | 63 | 25 | 172 | ScrF1 | Wt = 143 |
| N12R | 5′-tgg gta aag atg atc cga caa gtg a-3′ | 63 | 25 | 172 | ScrF1 | Mut = 172 |
| N13F | 5′-ctc cag aag tgt gag gcc gat-3′ | 63 | 25 | 250 | DpnII | Wt = 250 |
| N13R (MM) | 5′-ctg gat tgt cag tgc gct ttt ccc aag a-3′ | 63 | 25 | 250 | DpnII | Mut = 221 |
| N61F (MM) | 5′-tgt ttg ttg gac ata ctg gat aca gct gta-3′ | 63 | 25 | 225 | BsrG1 | Wt = 225 |
| N61R | 5′-tct tcc cta gtg tgg taa cct c-3′ | 63 | 25 | 225 | BsrG1 | Mut = 198 |
| K12F (MM) | 5′-gac tga ata taa act tgt ggt agt tgg acc t-3′ | 63 | 25 | 157 | ScrF1 | Wt = 127 |
| K12R | 5′-caa aga atg gtc ctg cac cag t-3′ | 63 | 25 | 157 | ScrF1 | Mut = 157 |
Method type/assay . | Primer sequence . | Annealing temperature, °C . | No. PCR cycles . | Amplicon size, bp . | RFLP enzyme . | RFLP digest product sizes, bp . |
|---|---|---|---|---|---|---|
| Mutation screening | ||||||
| DHPLC | ||||||
| K61F* | 5′-tgt gtt tct ccc ttc tca gga ttc-3′ | 60 | 35 | 158 | NA | NA |
| K61R | 5′-tgg caa ata cac aaa gaa agc c-3′ | 60 | 35 | 158 | NA | NA |
| K61F* | 5′-gat tcc tac cgg aag cag gt-3′ | 60 | 35 | 151 | NA | NA |
| K61R | 5′-tgg tgt tgt tga tgg caa ac-3′ | 60 | 35 | 151 | NA | NA |
| RFLP and sequencing | ||||||
| K12F (MM) | 5′-gac tga ata taa act tgt ggt agt tgg acc t-3′ | 56 | 35 | 121 | BstN1 (7.5 units) | Wt = 91 |
| K12R | 5′-aac aag att tac ctc tat tgt tgg atc a-3′ | 56 | 35 | 121 | (7.5 units) | Mut = 121 |
| H12F | 5′-agg aga ccc tgt agg agga-3′ | 60 | 35 | 170 | Msp1 (10 units) | Wt = 23, 56, 91 |
| H12R | 5′-gcg cta ggc tca cct cta t-3′ | 60 | 35 | 170 | (10 units) | Mut = 23, 147 |
| Mutant allele quantification: PCR with a end-labeled radioactive primer | ||||||
| N12F (MM) | 5′-act gag tac aaa ctg gtg gtg gtt gga cca-3′ | 63 | 25 | 172 | ScrF1 | Wt = 143 |
| N12R | 5′-tgg gta aag atg atc cga caa gtg a-3′ | 63 | 25 | 172 | ScrF1 | Mut = 172 |
| N13F | 5′-ctc cag aag tgt gag gcc gat-3′ | 63 | 25 | 250 | DpnII | Wt = 250 |
| N13R (MM) | 5′-ctg gat tgt cag tgc gct ttt ccc aag a-3′ | 63 | 25 | 250 | DpnII | Mut = 221 |
| N61F (MM) | 5′-tgt ttg ttg gac ata ctg gat aca gct gta-3′ | 63 | 25 | 225 | BsrG1 | Wt = 225 |
| N61R | 5′-tct tcc cta gtg tgg taa cct c-3′ | 63 | 25 | 225 | BsrG1 | Mut = 198 |
| K12F (MM) | 5′-gac tga ata taa act tgt ggt agt tgg acc t-3′ | 63 | 25 | 157 | ScrF1 | Wt = 127 |
| K12R | 5′-caa aga atg gtc ctg cac cag t-3′ | 63 | 25 | 157 | ScrF1 | Mut = 157 |
bp indicates base pair; F indicates forward; R, reverse; MM, mismatched; Wt, wild type; and Mut, mutant.
DHPLC temperature was 59°C for K61 and 64°C for H61.