| Popis: |
Single nucleotide polymorphisms (SNPs) can be difficult to detect using traditional PCR and gel electrophoresis, especially when no enzyme restriction sites overlap the SNP. Although alleles can be identified through amplification with flanking primers and sequencing, this adds time and cost, and sequencing is typically outsourced. Tetra-primer Amplification-Refractory Mutation System (ARMS)-PCR allows SNPs to be distinguished through a 3' primer mismatch at the SNP site for one allele, and an additional mismatch 1 - 3 bases upstream from the 3' end (Figure 1A), limiting amplification to only the allele with a single mismatch (Ye et al. 2001). Each internal primer (one forward and one reverse) is allele-specific, while external primers are different distances from the SNP, producing allele-specific fragments of different sizes in a single PCR reaction that can be distinguished by gel electrophoresis. This is useful for single-worm PCR where separate reactions may not be feasible, such as selection of individuals from a segregating population. Here, we designed primers and optimized PCR conditions to allow clear genotyping of lin-65(n3441), a GíA point mutation, without the need for sequencing. Additionally, we show that minor changes to reaction conditions allow for amplification of the larger flanking fragment (Figure 1B) which can then be gel purified and sequenced. While this may not be necessary for routine genotyping, sequencing the flanking fragment provides confirmation and higher confidence in SNP identification. To demonstrate the accuracy of our protocol, N2 and lin-65(n3441) adult hermaphrodites, a heterozygous F1 hermaphrodite, and twelve segregating F2 progeny were genotyped by PCR using conditions optimized for amplification of the flanking fragment (Figure 1C). Flanking fragments were then gel purified and sequenced. Results showed the presence of the G allele (WT) in N2, F2-7, and F2-10, the A allele (n3441) in lin-65(n3441) and F2-4, and heterozygous G/A in F1 and the remaining F2 samples, confirming the genotypes indicated by PCR. Interestingly, sequencing showed a higher proportion of the G allele in all nine heterozygous samples and the heterozygous control. Those samples, as well as the homozygous n3441 sample and control, were also heterozygous G/A at the -2 base, showing a higher proportion of the A allele; this change corresponds to the introduced GíA mismatch in the n3441-specific internal primer. These two observations suggest the possibility of low level overlapping PCR during amplification, incorporating the primer sequence into a portion of PCR product. Additionally, the higher proportion of the G allele at the SNP site indicates amplification bias of the n3441-specific primer over the flanking forward primer in the presence of the n3441 allele, leaving the two flanking primers with mostly the WT allele for amplification in heterozygotes. This idea is supported by the visibly brighter n3441 bands in all samples where the allele is present. |
