Design PCR primers for SNP genotyping
Back to ToolsSNP genotyping is the process of determining which DNA sequence variants (alleles) are present at specific SNP positions in an individual's genome. PCR primers are needed to amplify the region containing the SNP before genotyping methods like Sanger sequencing, RFLP analysis, or allele-specific PCR can be applied. Well-designed primers ensure efficient and specific amplification of the target region.
The SNP position should be specified as a 0-based index within your input sequence. For example, if your SNP is the 21st nucleotide in your input sequence, you would enter "20" as the SNP position (counting starts from 0). The tool will design primers that flank this position while optimizing for parameters like melting temperature and GC content.
For most SNP genotyping applications, primers between 18-25 nucleotides in length work well. This range provides a good balance between specificity (longer primers are more specific) and annealing efficiency (shorter primers anneal more easily). The default value of 20 nucleotides is suitable for most standard PCR conditions, but you can adjust based on your specific requirements.
The optimal amplicon length depends on your specific application. For standard PCR and Sanger sequencing, amplicons of 150-500 bp generally work well. For allele-specific PCR or RFLP analysis, you might prefer shorter amplicons (100-300 bp). For next-generation sequencing applications, even shorter amplicons (70-200 bp) may be preferred. The default value of 200 bp is a good starting point for most applications.
Tm (melting temperature) is the temperature at which half of the DNA strands are denatured. Primers with similar Tm values (within 5°C of each other) perform best in PCR. Ideally, primers should have Tm between 55-65°C. GC% refers to the percentage of G and C nucleotides in the primer. Primers with 40-60% GC content generally perform well, providing balanced binding strength and specificity. Extremely high or low GC content can lead to PCR problems.