Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics

Momčilo Gavrilov, Joshua Y. C. Yang, Roger S. Zou, Wen Ma, Chun-Ying Lee, Sonisilpa Mohapatra, Jimin Kang, Ting-Wei Liao, Sua Myong and Taekjip Ha ()
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Momčilo Gavrilov: Johns Hopkins University School of Medicine
Joshua Y. C. Yang: Johns Hopkins University School of Medicine
Roger S. Zou: Johns Hopkins University School of Medicine
Wen Ma: University of California
Chun-Ying Lee: Johns Hopkins University
Sonisilpa Mohapatra: Johns Hopkins University School of Medicine
Jimin Kang: Johns Hopkins University
Ting-Wei Liao: Johns Hopkins University
Sua Myong: Johns Hopkins University
Taekjip Ha: Johns Hopkins University School of Medicine

Nature Communications, 2022, vol. 13, issue 1, 1-14

Abstract: Abstract Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Date: 2022
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DOI: 10.1038/s41467-022-34076-0

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