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

Area of Science:

• Molecular Biology
• Genetic Engineering
• Biotechnology

Background:

• Accurate genetic testing relies on high-quality reference materials.
• Existing reference materials may not fully represent complex heterozygous genotypes.

Purpose of the Study:

• To develop a novel compound heterozygous reference material (hetRM) for Factor II (FII) G20210A and Factor V (FV) G1691A mutations.
• To utilize gene synthesis technology for precise construction of the hetRM.

Main Methods:

• Gene synthesis with inverted insertion of wild-type and mutant fragments into a cloning vector.
• Amplification in E. coli and verification using real-time PCR, hydrolysis probes, allele-specific amplification, reverse hybridization, and DNA sequencing.
• Assessment of stability over six months at -80°C.

Main Results:

• Successful preparation and verification of a compound heterozygous reference material (hetRM).
• All applied methods confirmed the accuracy of the hetRM.
• The hetRM demonstrated excellent stability with no significant changes in real-time PCR values after storage.

Conclusions:

• A novel, stable, and commutable genetic reference material for FII G20210A and FV G1691A testing was successfully prepared using gene synthesis.
• The hetRM is available in large quantities and wide concentration ranges, suitable for diagnostic applications.
• This approach provides a reliable tool for validating molecular diagnostic tests for these specific genetic mutations.