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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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Related Experiment Video

Updated: May 14, 2026

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
15:28

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

Published on: September 3, 2009

The primer extension assay.

Michael F Carey, Craig L Peterson, Stephen T Smale

    Cold Spring Harbor Protocols
    |February 5, 2013
    PubMed
    Summary
    This summary is machine-generated.

    The primer extension assay experimentally determines gene transcription start sites by identifying the 5' end of messenger RNA (mRNA). This method precisely maps the mRNA 5' end using radiolabeled complementary DNA (cDNA) synthesis and gel electrophoresis.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Biochemistry

    Background:

    • Accurate determination of transcription start sites (TSS) is crucial for understanding gene regulation.
    • Traditional methods may lack precision or require extensive optimization.

    Purpose of the Study:

    • To describe and validate the primer extension assay for precise TSS mapping.
    • To establish a reliable method for identifying the 5' end of messenger RNA (mRNA).

    Main Methods:

    • Utilizes a synthetic oligonucleotide primer complementary to mRNA, labeled with radioactive phosphorus ([γ-(32)P]ATP).
    • Primer extension is catalyzed by reverse transcriptase (RT) in the presence of deoxyribonucleoside triphosphates.
    • Radiolabeled complementary DNA (cDNA) products are analyzed via denaturing polyacrylamide gel electrophoresis and autoradiography.

    Main Results:

    • The assay generates radiolabeled cDNA products whose sizes correspond to the distance from the primer's 5' end to the mRNA's 5' end.
    • Analysis against a sequencing ladder or molecular weight standards allows precise size determination.
    • The transcription start site can be identified with single-nucleotide accuracy.

    Conclusions:

    • The primer extension assay is a highly accurate method for experimentally determining gene transcription start sites.
    • This technique provides a robust tool for molecular biologists studying gene expression and regulation.