Single Nucleotide Polymorphism (SNP) are point mutation that occur in more than 1% of the population. These genetic polymorphisms can be studied by first amplifying the target site using the polymerase Chain Reaction (PCR). Subsequently, variants can be determined, for example, by using restriction enzyme (RFLP) or by Sanger Sequencing. RFLP are limited by availability of restriction site at the target sequence and Sanger Sequencing can get expensive. High Resolution Melt Analysis (HRM) is another technique for SNP analysis. A method routinely performed in our laboratory using Qiagen Rotorgene Q and Qiagen Qiagility platform. It is versatile and cost effective. As a closed tube method, it is also less prone to post-PCR contamination and variation. In HRM analysis, the region of interest is amplified by PCR in the presence of a double stranded DNA (dsDNA) binding dye. This dye is highly fluorescent when bound to dsDNA. Next, the amplicons are gradually denatured by raising temperature in measured small increments. When dsDNA denatures, releasing the dsDNA dye, decrease in fluorescence are captured to plot a melt curve. As nucleic acid sequence influences the temperature at which dsDNA dissociate, a single nucleotide variant can be identified by their differing melt profile. HRM can also be used to classify SNP genotypes.Steps in SNP genotyping:
1) Extraction of DNA
2) Designing primers
3) Primer validation
4) PCR and HRM
5) Result analysis and report
1) SNP genotyping
We can provide analysis of SNP genotyping, from DNA extraction, Primer design to Melt curve analysis and result reporting. Partial service can also be provided. For an example, if ready DNA is available and only PCR/HRM analysis is needed.
2) QIAgility Automated liquid handler
The QIAgility is capable of dispensing liquid with precision and helps eliminate variation from manual pipetting. The QIAgility can be used to help setup ad-hoc experiments for example PCR, aliquot samples etc.
3) Rotorgene Q real-time PCR cycler (2 plex - Green, Yellow)
The Rotorgene Q rotary design enables for tube-to-tube thermal uniformity and eliminates the need for ROX reference dye. Rotogene Q is particularly suited for HRM analysis. It is used both for Real-time PCR and HRM experiments at our lab.
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