Efficient variant phasing utilizing a replication cycle reaction system

Area of Science:

• Genomics
• Molecular Biology
• Genetic Diagnostics

Background:

• Determining the cis or trans configuration of heterozygous variants in autosomal recessive genes is critical for genetic diagnosis.
• Existing methods like subcloning and droplet digital PCR have limitations in variant distance and technical feasibility.

Purpose of the Study:

• To develop and validate a novel method for phasing widely spaced heterozygous variants using replication cycle reaction (RCR).
• To overcome the distance limitations of current variant phasing techniques.

Main Methods:

• Circular DNA molecules were created by ligating CRISPR/Cas9-cleaved genomic fragments with an oriC-AmpR cassette.
• Replication cycle reaction (RCR) was used to amplify these circular DNA molecules.
• Reaction conditions were optimized, and phasing accuracy was validated using electrophoresis and Sanger sequencing.

Main Results:

• RCR successfully amplified genomic regions up to 104 kb, enabling phasing of heterozygous variants.
• Monoallelic amplification for straightforward phasing was achieved at lower gDNA-to-cassette ratios.
• The method confirmed compound heterozygosity in all 7 tested patients with variants up to 152 kb apart.

Conclusions:

• Replication cycle reaction (RCR) is an effective method for phasing multiple heterozygous variants across large genomic distances.
• This technique offers a significant advancement for genetic diagnostics, particularly for autosomal recessive diseases.