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Real Time RT-PCR02:57

Real Time RT-PCR

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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: Mar 12, 2026

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
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Library Quantification Using SYBR Green-Quantitative Polymerase Chain Reaction (qPCR).

Elaine Mardis, W Richard McCombie

    Cold Spring Harbor Protocols
    |November 3, 2016
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    Kapa Biosystems developed a new DNA polymerase for SYBR Green quantitative PCR (qPCR), improving the amplification of challenging DNA targets like GC-rich sequences for accurate library quantification.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genomics

    Background:

    • Accurate quantification of DNA libraries is crucial for downstream applications.
    • GC-rich DNA sequences and longer fragments pose amplification challenges for standard DNA polymerases in qPCR.
    • Existing methods may lack efficiency and robustness in complex DNA library analysis.

    Purpose of the Study:

    • To engineer a DNA polymerase optimized for SYBR Green-based quantitative PCR (qPCR).
    • To enhance the amplification efficiency of challenging DNA targets, including GC-rich sequences.
    • To provide a robust solution for complex DNA library quantification.

    Main Methods:

    • Engineering of a novel DNA polymerase with enhanced properties for qPCR.
    • Utilizing SYBR Green chemistry for real-time DNA amplification monitoring.
    • Testing the polymerase's performance on GC-rich DNA and longer DNA fragments.

    Main Results:

    • The engineered DNA polymerase demonstrates efficient amplification of GC-rich DNA targets.
    • Robust amplification of longer DNA fragments is achieved across a wide GC content range.
    • Kapa Library Quantification Kits incorporate this polymerase for improved performance.

    Conclusions:

    • The engineered DNA polymerase offers superior performance for SYBR Green-based qPCR.
    • This innovation enables more accurate quantification of complex DNA libraries.
    • Kapa Biosystems provides a robust tool for challenging DNA amplification and quantification needs.