DUPLICA Real Time MTHFR C677T Genotyping Kit From Genycell Biotech

The assay analyzes the C-to-T mutation occurring at nucleotide 677 of the gene MTHFR (Methylen-tetra-hydro-pholate reductase) involved in the re-methylation of homocysteine to methionine. This mutation leads to decreased enzymatic activity, with increased plasmatic levels of homocysteine which is thought to be a risk factor for cardiovascular diseases. A fluorogenic probe-based PCR assay is used for the detection of the point mutation. Two dye-labeled probes are used in this allelic discrimination assay: one probe for each allele in the two-allele system. Each probe consists of an oligonucleotide with a 5' reporter and a 3' quencher dye. FAM (6-carboxy-fluorescin) is covalently linked to the 5'-end of the probe for detection of the wild-type allele. HEX (hexachlorofluorescein, read also by the channel Cy3) is covalently linked to the 5'-end of the probe for detection of the mutant allele. Each of the reporters is quenched by TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) attached via a linker arm located at the 3' end of each probe. The quencher can only quench the reporter fluorescence when the two dyes are close to each other. This is only in the case of the intact probe. Once amplification occurs, the probe is degraded by the 5'-3' exonuclease activity of the Taq DNA polymerase and the fluorescence is detected by an optical system, and fluorescence increases as the DNA is amplified. This assay allows the direct detection of the specific PCR products by monitoring the increase in fluorescence of a dye-labeled oligonucleotide probe.

The kit contains all necessary reagents to perform 32 tests, including the wild-type and the mutated reference controls. I use it to detect a point mutation in 5 ul of genomic DNA isolated from 200 ul of whole blood. The assay is robust enough to allow the concentration of DNA does not need to be determined prior to running the assay. The preparation of the PCR mix is extremely easy: 10 ul of the amplification mix plus 10 ul of oligo mix (ready to use), and 5 ul of extracted DNA (200-500 ng). It's important to keep the samples cold until the run starts, to avoid non-specific priming and subsequent background. Bubbles in the PCR tubes should also be avoided since they can interfere with accurate fluorescence detection, thus leading to an aberrant fluorescence curve. The assay takes approximately 1 hour to run.

The results are interpreted as follows: the presence of fluorescence from the FAM probe means amplification of the wild type allele and the presence of fluorescence from the Hex-Cy3 probe means amplification of the mutant allele. Since FAM or Cy3 signals will not be completely missing in the mutated or wild-type samples, respectively, close attention has to be paid to read heterozygous samples.

It is possible to mix left over amounts of reagents belonging to the same lot number of the kits. In this case, the negative control may show a late fluorescence but it does not interfere with the interpretation of the results.