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Related Concept Videos

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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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

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Related Experiment Video

Updated: Jun 25, 2026

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
10:41

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Published on: March 29, 2017

Mutation detection by real-time PCR: a simple, robust and highly selective method.

John Morlan1, Joffre Baker, Dominick Sinicropi

  • 1Genomic Health, Inc., Redwood City, California, United States of America.

Plos One
|February 26, 2009
PubMed
Summary
This summary is machine-generated.

A new Allele-Specific PCR with Blocking reagent (ASB-PCR) method enables sensitive and selective detection of genetic mutations using real-time PCR. This robust assay works on various tissues, including formalin-fixed samples, simplifying molecular diagnostics.

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Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation
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Last Updated: Jun 25, 2026

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
10:41

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Published on: March 29, 2017

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
08:23

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Published on: September 25, 2018

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation
07:17

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

Published on: August 23, 2024

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Molecular tests, including gene expression and mutation analysis, are crucial for cancer diagnosis, prognosis, and treatment selection.
  • Current mutation detection assays often require extensive optimization and are not always compatible with real-time PCR.
  • There is a need for efficient, selective, and real-time PCR-compatible mutation detection methods.

Purpose of the Study:

  • To develop a real-time PCR-based mutation assay methodology.
  • To create a method that is highly selective and compatible with real-time PCR.
  • To address the limitations of existing mutation detection assays.

Main Methods:

  • Utilized TaqMan real-time PCR technology combined with Allele-Specific PCR and a Blocking reagent (ASB-PCR).
  • Developed a set of reagent design rules for sensitive and selective detection of mutations.
  • Applied the method to various tissue types, including formalin-fixed paraffin-embedded specimens.

Main Results:

  • ASB-PCR effectively suppresses amplification of the wild-type allele, enabling detection of mutations against a large excess of wild-type DNA/RNA.
  • Achieved sensitive and selective detection of single point substitutions, insertions, or deletions.
  • Demonstrated compatibility with both DNA and RNA from diverse tissue sources.

Conclusions:

  • ASB-PCR is a simple, robust, and highly selective method for single nucleotide mutation and polymorphism detection.
  • The developed design rules consistently yield effective mutation assays without extensive redesign.
  • The method is compatible with formalin-fixed tissues and simultaneous gene expression analysis, making it accessible to standard research labs.