In:
PLOS ONE, Public Library of Science (PLoS), Vol. 15, No. 12 ( 2020-12-31), p. e0244464-
Abstract:
Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 10 7 , drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases’ behaviors and functions in daily genetic engineering experiments.
Type of Medium:
Online Resource
ISSN:
1932-6203
DOI:
10.1371/journal.pone.0244464
DOI:
10.1371/journal.pone.0244464.g001
DOI:
10.1371/journal.pone.0244464.g002
DOI:
10.1371/journal.pone.0244464.g003
DOI:
10.1371/journal.pone.0244464.g004
DOI:
10.1371/journal.pone.0244464.t001
DOI:
10.1371/journal.pone.0244464.s001
DOI:
10.1371/journal.pone.0244464.s002
DOI:
10.1371/journal.pone.0244464.s003
DOI:
10.1371/journal.pone.0244464.s004
DOI:
10.1371/journal.pone.0244464.s005
DOI:
10.1371/journal.pone.0244464.s006
DOI:
10.1371/journal.pone.0244464.s007
DOI:
10.1371/journal.pone.0244464.s008
DOI:
10.1371/journal.pone.0244464.s009
DOI:
10.1371/journal.pone.0244464.s010
DOI:
10.1371/journal.pone.0244464.s011
DOI:
10.1371/journal.pone.0244464.s012
DOI:
10.1371/journal.pone.0244464.s013
DOI:
10.1371/journal.pone.0244464.s014
DOI:
10.1371/journal.pone.0244464.s015
DOI:
10.1371/journal.pone.0244464.s016
DOI:
10.1371/journal.pone.0244464.r001
DOI:
10.1371/journal.pone.0244464.r002
DOI:
10.1371/journal.pone.0244464.r003
DOI:
10.1371/journal.pone.0244464.r004
Language:
English
Publisher:
Public Library of Science (PLoS)
Publication Date:
2020
detail.hit.zdb_id:
2267670-3
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