A fusion of Taq DNA polymerase with the CL7 protein from Escherichia coli remarkably improves DNA amplification
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.
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.
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.
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Posted 10 Jan, 2021
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A fusion of Taq DNA polymerase with the CL7 protein from Escherichia coli remarkably improves DNA amplification
Posted 10 Jan, 2021
On 10 Jan, 2021
On 07 Jan, 2021
On 07 Jan, 2021
On 07 Jan, 2021
Posted 18 Dec, 2020
Received 18 Dec, 2020
On 13 Dec, 2020
On 11 Dec, 2020
On 10 Dec, 2020
Invitations sent on 10 Dec, 2020
On 10 Dec, 2020
On 10 Dec, 2020
Posted 20 Oct, 2020
Received 08 Nov, 2020
On 08 Nov, 2020
Received 06 Nov, 2020
Received 24 Oct, 2020
On 20 Oct, 2020
On 18 Oct, 2020
On 16 Oct, 2020
Invitations sent on 16 Oct, 2020
On 16 Oct, 2020
On 15 Oct, 2020
On 15 Oct, 2020
On 09 Oct, 2020
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.
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.
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