Method for detection of GSTM1, GSTT1 deletion variantsusing Taq 2x Dual master mix / Монголын Анагаах Ухаан

ABSTRACT

INTRODUCTION:GSTs are a family of antioxidant enzymes that responsible for the detoxification of many carcinogens.Glutathione S-transferases are polymorphic in humans and the null genotypes are results in lack ofenzyme activity.In many studies the polymorphisms of GSTM1, GSTT1 have been associated withcancers of the lung, bladder, breast and colon.GOAL:In this research we aimed to establish PCR condition for obtaining “long” PCR product for detectionof deletions in GSTT1, GSTM1 genes using various master mixes, which would help us further todetect heterozygous variants for these two genes in Mongolian population.MATERIALS AND METHODS:Three kinds of commercial master mixes as Go Taq PCR master mix (USA), Taq 2x Dual master mix(Mongolia), and DyNAzyme EXT buffer were tested at various PCR conditions on 117 DNA samples,isolated in three ways such as phenol chloroform extraction method, guanidine hydrochloride methodand using Promega Wizard Genomic Fragment DNA Extraction Kit from fresh blood lymphocytes,buccal swabs and dried blood spots.RESULTS:Three types of samples were used for DNA extraction such as buccal swabs, dried onto soft tissueblood spots and fresh peripheral blood lymphocytes, using three kind extraction methods from whichDNA template obtained from fresh blood isolated by guanidine chloride method had best quality.Combination as template DNA from fresh blood, guanidine chloride DNA extraction method and Taq2x Dual master mix (Mongolia) resulted in all four band, whereas other combination did not displaydesired results.CONCLUSIONS:Out of three kinds commercial master mixes tested in this study for various PCR templateDNApreparation and PCR conditions we observed that:1. PCR with Taq 2x Dual master mix (Mongolia) resulted in all four initially desiredPCR productsas 625bp for GSTM1, 969bp for GSTT1 genes and 4748bp for GSTM1, 3106bp for GSTT1 genedeletions correspondingly;2. Template genome DNA prepared from fresh peripheral blood lymphocytes by guanidinehydrochloride extraction methods suited best for “long” PCR reaction;3. Using Taq 2x Dual master mix produced in Mongolia saved us time and was cheaper.4. Multplex primer mix is excellent tool in research of GST gene polymorphism.