Nový koncept přípravy referenčních materiálů s heterozygotním genotypem pro molekulárně diagnostické účely
Using the technique of enzymatic gene assembly we developed a compound heterozygous reference material for analysis of G20210A mutation in the Factor II and G1691A in the Factor V genes. The prepared clone contained both wild-type and mutant fragments of the genes. The DNA fragments carrying the mutations were inserted into the cloning vector in the inverse orientation relative to the wild-type ones. The clone performance was verified by DNA sequencing and by the allele discrimination real-time PCR methods. Our novel approach makes it possible to prepare a compound heterozygous reference material for any rare variants at biallelic polymorphic sites if analyzed by real-time PCR or other methods.