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Related Concept Videos

PCR01:32

PCR

Overview
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
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Optimizing PCR assays for DNA-based cancer diagnostics.

Ali Bashir1, Qing Lu, Dennis Carson

  • 1Department of Computer Science, University of California, San Diego, La Jolla, California 92129, USA.

Journal of Computational Biology : a Journal of Computational Molecular Cell Biology
|April 10, 2010
PubMed
Summary

This study introduces a new algorithm for designing PCR primers and probes to detect cancer DNA rearrangements. The method enables accurate detection of these mutations even with mixed normal and tumor cells, aiding early cancer diagnosis.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Somatic DNA rearrangements are common in cancers but difficult to detect due to mixed normal/tumor cells and variable breakpoint locations.
  • Current diagnostic methods struggle with the high background of normal DNA and the lack of conserved rearrangement boundaries across individuals.

Purpose of the Study:

  • To develop a robust algorithm for designing polymerase chain reaction (PCR) primers and oligonucleotide probes to accurately assay variant DNA rearrangements in cancer.
  • To overcome challenges in detecting cancer-specific mutations admixed with normal DNA and account for variable breakpoint locations.

Main Methods:

  • Developed a combinatorial optimization algorithm for designing PCR primers and probes that tile entire genomic regions around rearrangements.
  • Implemented a novel alternating multiplexing strategy for efficient and sensitive detection of amplified mutant DNA.
  • Validated the technique using simulations and applied it to assay genomic lesions in cancer cell lines with CDKN2A locus disruption.

Main Results:

  • Simulations demonstrated near-optimal detection capabilities, even for non-symmetric genomic regions.
  • The alternating multiplexing strategy was proven optimal for breakpoint detection.
  • Successfully detected variable CDKN2A locus breakpoints in multiple cancer cell lines, even after multiple rearrangements.

Conclusions:

  • The developed algorithm and multiplexing strategy provide a successful protocol for detecting variant DNA rearrangements in cancer.
  • This approach facilitates early diagnosis and monitoring of cancer by enabling robust detection of key genomic lesions.