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Real Time RT-PCR02:57

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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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Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
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Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs.

Hyo Yong Kim1, Taihua Li2, Cheulhee Jung1

  • 1Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST 291 Daehak-ro, Yuseong-gu Daejeon 305-701 Republic of Korea hgpark@kaist.ac.kr +82-42-350-3910 +82-42-350-3932.

RSC Advances
|May 13, 2022
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Summary
This summary is machine-generated.

This study introduces novel quantitative PCR (qPCR) methods using fluorescent nucleobase analogs for sensitive and cost-effective detection of nucleic acids, including those from sexually transmitted disease pathogens.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Nucleic Acid Chemistry

Background:

  • Quantitative PCR (qPCR) is crucial for nucleic acid detection but faces limitations in specificity and cost.
  • Existing methods like SYBR Green and TaqMan probes have drawbacks.
  • Fluorescent nucleobase analogs offer potential for novel detection strategies.

Purpose of the Study:

  • To develop novel qPCR methods utilizing fluorescent nucleobase analogs.
  • To create both signal-off and signal-on qPCR detection systems.
  • To improve specificity and reduce assay costs compared to existing qPCR methods.

Main Methods:

  • Developed a signal-off qPCR method using primers with pyrrolo-dC (PdC) fluorescent nucleobase analogs.
  • Developed a signal-on qPCR method using primers with 5'-overhangs and PdC-incorporated DNA probes.
  • Utilized conformation-dependent fluorescence and probe detachment mechanisms for signal generation.

Main Results:

  • Demonstrated successful quantification of target nucleic acids from sexually transmitted disease pathogens with high accuracy.
  • Achieved reduced fluorescence in signal-off mode due to PdC incorporation into primers.
  • Observed enhanced fluorescence in signal-on mode via probe detachment, enabling multiplex detection with a single probe.

Conclusions:

  • The novel qPCR methods offer improved specificity and reduced assay costs.
  • The signal-on method allows for cost-effective multiplex detection of multiple targets.
  • These strategies provide advanced alternatives to current SYBR Green and TaqMan probe-based qPCR assays.