Genetics

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Reagent kit is intended for differential determination of single-nucleotide polymorphisms of the coagulation system and folate cycle by real-time PCR with detection of melting curves. The kits contain reagents for real-time PCR only.

“Molgen Genetics Haemostatic Disease (MTR/MTRR/MTHFR) Kit” is intended for differential determination of 4 single nucleotide polymorphisms (MTR:2756 A/G; MTRR:66 A/G; MTHFR:677 С/T and MTHFR:1298 А/C) in genes of the human folate cycle by real-time polymerase chain reaction (PCR) followed by melting of hybridisation products with a fluorescent tag and detection of melting curves.

The kit is intended for use with block-type PCR cycler CFX96 (Bio-Rad, USA) such as Adaltis AmpliLab.

The assay kit can be applied in clinical practice for a comprehensive study of the genetic factors which increase the risk of disorders in the folate cycle.

The extraction of human DNA from clinical samples (buccal epithelium, whole blood) is performed using the kit “MOLgen Universal Extraction Kit”.

The kit is designed for the analysis of 48 samples, including control samples.

The principle of the method is based on the amplification of the selected human DNA region followed by detection of melting curves of hybrid complexes in PCR products and specific probes. The process of amplification consists in repeated cycles: temperature denaturation of DNA matrix, annealing primers with complementary sequences of DNA matrix, and synthesis of a complementary chain from these primers with Taq polymerase.

After the amplification reaction the lowering of temperature results in hybridisation of the specific DNA probe with the amplification product and in decrease of a fluorescent signal in the tube. The release of DNA probe bearing a fluorescent dye is occurred during the temperature melting of formed duplexes. In case of incomplete complementarity of DNA probe and the selected DNA region, the melting temperature of the duplex is lower than that in case of complete complementarity. Thus, the melting temperature of probe duplexes and amplification products is different for two allelic variants of a gene. As a result, the genotype (normal homozygote, heterozygote, mutant homozygote) can be identified for each of the polymorphisms to be determined.

For testing of each sample, two ready-to-use master mixes RMM1 and RMM2 are used. In each of RMMs DNA amplification and detection of melting curves is performed for determination of two genetic polymorphisms.

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