DNA replication complexes (replisomes) routinely encounter proteins and unusual nucleic acid structures that can impede their progress. Barriers can include transcription complexes and R‐loops that form when RNA hybridizes with complementary DNA templates behind RNA polymerases. Cells encode several RNA polymerase and R‐loop clearance mechanisms to limit replisome exposure to these potential obstructions. One such mechanism is hydrolysis of R‐loops by ribonuclease HI (RNase HI). Here, we examine the cellular role of the interaction between
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.
more » « less- NSF-PAR ID:
- 10376657
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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