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

• DOI: 10.1038/s41467-022-34076-0
• Publication Date: 2022-10-23T08:02:48Z
• Authors: Momčilo Gavrilov; Joshua Y. C. Yang; Roger S. Zou; Wen Ma; Chun-Ying Lee; Sonisilpa Mohapatra; Jimin Kang; Ting-Wei Liao; Sua Myong; Taekjip Ha
• Keywords: Processivity; Amplicon; Multiple displacement amplification; genomic DNA; Recombinase Polymerase Amplification

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