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Overview
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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.
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Updated: Sep 1, 2025

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
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Critical insight into recombinase polymerase amplification technology.

Mustafa Ahmad Munawar1

  • 1Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland.

Expert Review of Molecular Diagnostics
|August 11, 2022
PubMed
Summary

Recombinase polymerase amplification (RPA) offers rapid nucleic acid amplification for point-of-care testing. Further research is needed to address RPA

Keywords:
Recombinase polymerase amplificationinhibitorsnonspecificitypoint-of-care testing

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Diagnostics

Background:

  • Recombinase polymerase amplification (RPA) is an emerging nucleic acid amplification technology.
  • RPA is suitable for point-of-care testing (POCT) and field applications due to its speed and compatibility with portable instruments and microfluidics.
  • Limited research has focused on the underlying chemistry, kinetics, and optimization of RPA, hindering its full potential.

Purpose of the Study:

  • To provide a comprehensive review of RPA technology, covering its molecular mechanisms, kit formats, and optimization strategies.
  • To critically analyze the applications, advantages, and disadvantages of RPA, with a focus on pathogen detection.
  • To highlight the critical issue of RPA nonspecificity and emphasize the need for further research to resolve it.

Main Methods:

  • Review of existing literature on RPA technology.
  • Analysis of RPA's molecular mechanism and reaction kinetics.
  • Discussion of RPA's compatibility with various detection assays and microfluidic devices.

Main Results:

  • RPA demonstrates significant potential for rapid pathogen detection, particularly in resource-limited settings.
  • The review identifies nonspecificity as a key challenge requiring further investigation and resolution.
  • RPA kinetics are discussed in relation to target length, product yield, and sensitivity.

Conclusions:

  • RPA is a promising alternative to PCR for various applications, especially in diagnostics.
  • Addressing RPA's shortcomings, particularly nonspecificity, and improving reagent accessibility are crucial for its widespread adoption.
  • Further research into RPA's fundamental chemistry and kinetics is essential for technological advancement.