A New Concept in Preparation of Reference Materials with Heterozygous Genotype for Molecular Diagnostic Purposes
Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine and Faculty Hospital, Hradec Králové
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.
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