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Real Time RT-PCR

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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Related Experiment Video

Updated: May 2, 2026

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

Published on: April 11, 2016

ACB-PCR quantification of somatic oncomutation.

Meagan B Myers¹, Page B McKinzie, Yiying Wang

¹Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, HFT-120, Jefferson, AR, 72079, USA, Meagan.Myers@fda.hhs.gov.

Methods in Molecular Biology (Clifton, N.J.)

|March 14, 2014

Summary

This summary is machine-generated.

Allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) enables sensitive detection of rare cancer-associated mutations. This method accurately quantifies mutant DNA at low levels, aiding in cancer risk assessment and diagnosis.

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

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Published on: March 29, 2017

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Last Updated: May 2, 2026

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

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

Area of Science:

Molecular Biology
Genetics
Oncology

Background:

Hotspot point mutations (oncomutations) in cancer genes are crucial biomarkers for cancer risk.
Allele-specific amplification methods are needed for sensitive detection of rare mutant alleles.
Quantitative detection of mutations is vital for cancer diagnosis, treatment, and risk assessment.

Purpose of the Study:

To describe the Allele-Specific Competitive Blocker-Polymerase Chain Reaction (ACB-PCR) methodology.
To demonstrate the utility of ACB-PCR for quantifying rare mutant DNA alleles.
To illustrate the application of ACB-PCR in measuring a specific KRAS mutation.

Main Methods:

ACB-PCR utilizes mutant-specific primers and a blocker primer to selectively amplify rare mutant DNA.
The blocker primer, with a non-extendable 3'-end, suppresses amplification of wild-type DNA.
Quantification is achieved by comparing unknown samples to mutant fraction (MF) standards.

Main Results:

ACB-PCR can quantify single basepair substitution mutations at mutant:wild type ratios as low as 10(-5).
The method demonstrated high sensitivity and specificity in detecting rare mutant alleles.
The chapter details the successful application of ACB-PCR for measuring the human KRAS codon 12 GGT to GAT mutation.

Conclusions:

ACB-PCR is a sensitive and quantitative method for detecting rare oncomutations.
This technique has significant applications in cancer risk evaluation, diagnosis, and treatment.
ACB-PCR provides a robust tool for precise measurement of specific cancer-related genetic alterations.