Preparing compound heterozygous reference material using gene synthesis technology: a model of thrombophilic mutations
Abstract
The aim of our study is to present a novel approach for preparing a compound heterozygous reference material (hetRM) using gene synthesis technology with inverted insertion of wild-type and mutant fragments into a single cloning vector. Factor II (G20210A) and Factor V (G1691A Leiden) gene mutations were used as an experimental model.
During the gene synthesis, DNA fragments were aligned in the following order: G1691 FV wild-type forward strain, G20210 FII wild-type forward strain, 1691A FV mutant reverse strain, 20210A FII mutant reverse strain. The complete chain was inserted into a pIDT SMART cloning vector and amplified in an E. coli competent strain.
For assessing hetRM characteristics and commutability, we used real-time PCR with subsequent melting curve analysis, real-time PCR with hydrolysis probes, allele-specific amplification, reverse hybridization, and dideoxynucleotide DNA sequencing. All five methods yielded concordant results of DNA analysis of the hetRM.
Differences in real-time PCR cycle threshold values after six-months of storage at -80 oC were not statistically significant from those obtained from freshly prepared hetRM aliquots, which is a good indication of their stability. By applying the procedures of gene synthesis and cloning technology, we prepared and verified a modelgenetic reference material for FII G20210A and FV G1691A testing with a compound heterozygous genotype.
The hetRM was stable, commutable, and available in large quantities and in a wide concentration range.