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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Published on: March 30, 2015

Novel applications of polymerase chain reaction to urinary nucleic acid analysis

Anatoly V Lichtenstein1, Hovsep S Melkonyan, L David Tomei

  • 1Cancer Research Center, Moscow, Russia.

Methods in Molecular Biology (Clifton, N.J.)
|August 19, 2006
PubMed

Insights

Transrenal DNA, fragments from dead cells, are found in urine. Optimizing DNA isolation and polymerase chain reaction (PCR) methods by reducing amplicon size enhances sensitivity for detecting mutations, even with excess wild-type DNA.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Cellular debris, including DNA fragments, circulates in the bloodstream.
  • These DNA fragments, termed Transrenal DNA (tDNA), can cross the kidney barrier and be detected in urine.
  • The low molecular weight of tDNA (150-200 bp) presents unique challenges for isolation and analysis.

Purpose of the Study:

  • To investigate the characteristics of Transrenal DNA (tDNA) in urine.
  • To explore optimized methods for isolating and analyzing tDNA, particularly for mutation detection.
  • To address the challenge of detecting rare mutations in the presence of abundant wild-type DNA.

Main Methods:

  • Characterization of Transrenal DNA (tDNA) size in urine.
  • Application of polymerase chain reaction (PCR) with reduced amplicon size for enhanced sensitivity.
  • Development and application of enriched PCR and stencil-aided mutation analysis for selective wild-type DNA elimination.

Main Results:

  • Transrenal DNA (tDNA) fragments of 150-200 bp are present in urine.
  • Reducing amplicon size in PCR significantly enhances the sensitivity of specific sequence detection.
  • Enriched PCR and stencil-aided mutation analysis effectively overcome the challenge of wild-type allele excess for mutation detection.

Conclusions:

  • Urine is a viable source for Transrenal DNA (tDNA) analysis.
  • Optimizing PCR conditions, specifically by reducing amplicon size, is crucial for sensitive tDNA detection.
  • Novel methods like enriched PCR and stencil-aided analysis enable robust detection of DNA mutations in tDNA samples.

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