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PCR01:32

PCR

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Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications.

Lorène Aeschbach1, Vincent Dion2

  • 1Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, 1015, Lausanne, Switzerland.

Scientific Reports
|December 23, 2017
PubMed
Summary

Researchers developed a new PCR method using Uracil N-Glycosylase (Ung) and dUTP to improve the amplification of expanded CAG/CTG repeats, reducing contamination risk in diagnosing neurological disorders.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Neurology

Background:

  • Expanded CAG/CTG repeats are the cause of 14 neurological and neuromuscular disorders.
  • Repeat tract size correlates with disease severity, and somatic instability affects disease progression.
  • Current PCR methods for analyzing these repeats suffer from low yields, increasing contamination risk.

Purpose of the Study:

  • To reduce carry-over contamination risk in PCR amplification of expanded CAG/CTG repeats.
  • To optimize protocols for sequencing, cloning, and instability analysis of these repeats.
  • To adapt existing methods for clinical diagnostics of repeat expansion disorders.

Main Methods:

  • Pre-treating samples with Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCR.
  • Applying Ung and dUTP PCR to amplification, sequencing, and cloning of expanded repeats.
  • Optimizing small-pool PCR (SP-PCR) for use with Ung/dUTP protocols without compromising data quality.

Main Results:

  • Successful PCR amplification, sequencing, and cloning of expanded CAG/CTG repeats using the Ung/dUTP method.
  • Optimized SP-PCR demonstrated applicability to large expansions (up to 1000 repeats) in myotonic dystrophy samples.
  • The new protocols maintain data quality while significantly reducing contamination risk.

Conclusions:

  • The developed Ung/dUTP PCR protocols effectively reduce contamination risk in analyzing expanded CAG/CTG repeats.
  • Optimized SP-PCR is suitable for clinical samples with large repeat expansions.
  • These protocols are expected to benefit molecular diagnostics for expanded repeat disorders.