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Research article
Wang Yaping
Hubei University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a double-stranded DNA binding-like protein from Escherichia coli.
Results
The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 aM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required.
Conclusions
Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.
Keywords
Taq DNA polymerase, CL7, fusion DNA polymerase, PCR amplification
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CURRENT STATUS: Under Review
Version (no preprint)
Posted 10 Jan, 2021
Editorial decision: Minor revision
On 10 Jan, 2021
Editor assigned
On 07 Jan, 2021
Submission checks complete
On 07 Jan, 2021
Editor invited
On 07 Jan, 2021
This version was not preprinted
Version (no preprint)
Posted 18 Dec, 2020
Review #1 received
Received 18 Dec, 2020
Reviewer #2 agreed
On 13 Dec, 2020
Reviewer #1 agreed
On 11 Dec, 2020
Editor assigned
On 10 Dec, 2020
Reviewers invited
Invitations sent on 10 Dec, 2020
Submission checks complete
On 10 Dec, 2020
Editor invited
On 10 Dec, 2020
This version was not preprinted
Version 1
Posted 20 Oct, 2020
No community comments so far
Review #3 received
Received 08 Nov, 2020
Editorial decision: Major revision
On 08 Nov, 2020
Review #2 received
Received 06 Nov, 2020
Review #1 received
Received 24 Oct, 2020
Reviewer #3 agreed
On 20 Oct, 2020
Reviewer #2 agreed
On 18 Oct, 2020
Editor assigned
On 16 Oct, 2020
Reviewers invited
Invitations sent on 16 Oct, 2020
Reviewer #1 agreed
On 16 Oct, 2020
Submission checks complete
On 15 Oct, 2020
Editor invited
On 15 Oct, 2020
First submitted
On 09 Oct, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
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This preprint is under consideration at BMC Biotechnology. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Wang Yaping
Hubei University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version (no preprint)
Posted 10 Jan, 2021
Editorial decision: Minor revision
On 10 Jan, 2021
Editor assigned
On 07 Jan, 2021
Submission checks complete
On 07 Jan, 2021
Editor invited
On 07 Jan, 2021
This version was not preprinted
Version (no preprint)
Posted 18 Dec, 2020
Review #1 received
Received 18 Dec, 2020
Reviewer #2 agreed
On 13 Dec, 2020
Reviewer #1 agreed
On 11 Dec, 2020
Editor assigned
On 10 Dec, 2020
Reviewers invited
Invitations sent on 10 Dec, 2020
Submission checks complete
On 10 Dec, 2020
Editor invited
On 10 Dec, 2020
This version was not preprinted
Version 1
Posted 20 Oct, 2020
No community comments so far
Review #3 received
Received 08 Nov, 2020
Editorial decision: Major revision
On 08 Nov, 2020
Review #2 received
Received 06 Nov, 2020
Review #1 received
Received 24 Oct, 2020
Reviewer #3 agreed
On 20 Oct, 2020
Reviewer #2 agreed
On 18 Oct, 2020
Editor assigned
On 16 Oct, 2020
Reviewers invited
Invitations sent on 16 Oct, 2020
Reviewer #1 agreed
On 16 Oct, 2020
Submission checks complete
On 15 Oct, 2020
Editor invited
On 15 Oct, 2020
First submitted
On 09 Oct, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a double-stranded DNA binding-like protein from Escherichia coli.
Results
The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 aM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required.
Conclusions
Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
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