Preparing compound heterozygous reference material using gene synthesis technology: a model of thrombophilic mutations

Abstract

Aims. 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. Methods. 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. Result. 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 °C were not statistically significant from those obtained from freshly prepared hetRM aliquots, which is a good indication of their stability. Conclusion. By applying the procedures of gene synthesis and cloning technology, we prepared and verified a model genetic 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.

Development of a new genetic reference material system based on Saccharomyces cerevisiae cells

“…6 A series of RMs are used according to the experimental conditions: 7 commercial cell lines and DNA samples; remaining patient samples; sample sharing between laboratories; remaining samples of published research results; 8 cell lines containing genetic mutations; 9,10 genetically modified cell lines; 11 and plasmids, PCR products, and synthetic DNA. 12 Many types of genetic detection of RMs have their own advantages and limitations. Clinical patient samples have limitations such as inconvenience, low numbers, poor homogeneity, and inability to prepare RMs in large quantities; 13 the process of constructing cell lines with genetic mutations is cumbersome; and the double-copy characteristic of human cell genomes causes the possibility of heterozygous mutations.…”