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

• Published in: Biomedical papers of the Medical Faculty of the University Palacký, Olomouc, Czechoslovakia Vol. 158; no. 4; pp. 539 - 543
• Main Authors: Beranek, Martin, Drastikova, Monika, Dulicek, Petr, Palicka, Vladimir
• Format: Journal Article
• Language: English
• Published: Czech Republic 01-12-2014
• Subjects: Cloning, Organism - methods; Escherichia coli; Factor V - genetics; Genetic Techniques; Mutation; Prothrombin - genetics; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Thrombophilia - genetics
• ISSN: 1213-8118; 1804-7521
• DOI: 10.5507/bp.2014.041

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 °C 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 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.