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Long-Range Polymerase Chain Reaction.

Ping Siu Kee1, Harsheni Karunanathie2, Simran D S Maggo1,3

  • 1Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.

Methods in Molecular Biology (Clifton, N.J.)
|August 22, 2023
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Summary

Long-range polymerase chain reaction (PCR) successfully amplifies large DNA fragments up to 20 kb. PCR enhancers are crucial for amplifying difficult DNA targets, improving long-range PCR efficiency.

Keywords:
Agarose gel electrophoresisDNA polymeraseLong ampliconsLong-range polymerase chain reaction (PCR)PCR additivesPCR enhancersPharmacogeneticsPrimer designProofreading enzymeThermal cycling

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Area of Science:

  • Molecular Biology
  • Genetics

Background:

  • Polymerase chain reaction (PCR) is a standard laboratory technique for DNA amplification.
  • Long-range PCR is a specialized method designed to amplify extensive DNA fragments.

Purpose of the Study:

  • To adapt and optimize a long-range PCR protocol for amplifying large DNA fragments from human genomic DNA.
  • To evaluate the efficacy of PCR enhancers in improving the success rate of long-range PCR.

Main Methods:

  • Utilized an adapted long-range PCR protocol.
  • Amplified DNA fragments of varying sizes (6.6, 7.2, 13, and 20 kb) from human genomic DNA.
  • Assessed the impact of PCR enhancers on amplification success.

Main Results:

  • Successfully generated PCR products ranging from 6.6 kb to 20 kb.
  • Demonstrated that PCR enhancers are necessary for the successful amplification of certain long DNA fragments.
  • Provided data on the effectiveness of specific enhancers in long-range PCR.

Conclusions:

  • The adapted long-range PCR protocol is effective for amplifying large DNA fragments.
  • PCR enhancers significantly improve the success and efficiency of long-range PCR, particularly for challenging targets.