Not all samples are amenable to genetic testing using the same methods. To ensure you receive the most accurate results we employ a number of methods as appropriate for your samples, including:
Melt curve analysis
Assays are designed to amplify specific regions of the target sequence in the presence of SYBR green. Post amplification, the amplicons are subjected to an increasing temperature while data is collected. SYBR green intercalates with double stranded DNA and releases light at a wavelength detected by the instrument. As the temperature increases the double stranded DNA will melt apart based primarily on two factors.
- The length of the DNA amplicon. The longer the amplicon the more hydrogen bonds present and the greater the required energy to separate the strands.
- The G-C content.
This is due to the fact that G-C pairs share 3 hydrogen bonds, while A-T pairings only share 2 hydrogen bonds. Therefore, amplicons designed for this method can be differentiated based on the melt profiles of these different products.
We utilize a multiplexed reaction containing the primers and probes designed for a specific region of the target and the primers and probes for a housekeeping gene which is present in the wild type genome. The 5′-3′ exonuclease activity of Taq Polymerase cleaves the probe in a hydrolysis reaction releasing the reporter fluorophore and the quencher. No longer being quenched, the fluorophore emits light at a wavelength detectable by the instrument. Increasing amounts of light signal increasing accumulation of the product. See the Wikipedia entry on TaqMan for more details.
The Kompetitive Allele Specific PCR genotyping system (KASP™) is a homogeneous, fluorescent, endpoint genotyping technology offered by LGC Genomics. KASP uses three components: test DNA with the SNP of interest; KASP Assay mix containing two different, allele-specific, competing forward primers with unique tail sequences and one reverse primer; the KASP Master mix containing FRET cassette plus Taq polymerase in an optimised buffer solution. KASP assays are designed by LGC Genomics. Primer sequences are not provided for any KASP assay.
While not our preferred method, sometimes post-PCR gel electrophoresis is the best method for some targets. Amplification is confirmed by melt curve analysis prior to running the amplicons on Agarose gels.