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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins
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Efficient variant phasing utilizing a replication cycle reaction system.

Akihiko Mitsutake1,2, Hiroyuki Ishiura1,3, Takashi Matsukawa1

  • 1Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Genetics in Medicine Open
|November 10, 2025
PubMed
Summary

Replication cycle reaction (RCR) phases heterozygous variants across large genomic distances, overcoming limitations of other methods. This technique is crucial for diagnosing genetic diseases by determining variant locations.

Keywords:
Autosomal recessive inheritanceCompound heterozygosityReplication cycle reactionVariant phasing

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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.