Mutant for improving Taq DNA polymerase tolerance and preparation method and application thereof
A polymerase and mutant technology, applied in the biological field, can solve the problems of low DNA polymerase activity and low tolerance, and achieve the effect of improving amplification ability and tolerance
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[0049] A method for preparing a mutant that improves Taq DNA polymerase tolerance, comprising the steps of:
[0050] Using the gene expression sequence of Taq DNA polymerase as the amplification template, and adding point mutation primers to perform point mutation PCR amplification to obtain the target gene expression sequence; wherein, the protein encoded by the target gene expression sequence is the same as the wild-type Compared with Taq DNA polymerase, it has S623N, R704S, A705L mutations. The target gene expression sequence is transformed into a host cell, and the transformed host cell is induced to express to obtain the mutant with improved Taq DNA polymerase tolerance.
[0051] In some embodiments of the present invention, the point mutation primers include a forward primer and a reverse primer, and the sequence of the forward primer is:
[0052] (hereinafter referred to as SEQ3),
[0053] The sequence of the reverse primer is:
[0054] (hereinafter referred to a...
Embodiment 1
[0065] Example 1: Construction of an expression vector of a Taq DNA polymerase mutant and its expression and purification. The specific process is as follows:
[0066] Using the plasmid containing the Taq enzyme gene derived from Escherichia coli as a template, first use the synthetic amplification primers SEQ3 and SEQ4 to carry out PCR amplification, after amplification, use DpnI enzyme to digest the original master plate, and the product of PCR amplification, namely The plasmid containing the mutated enzyme gene was transformed into competent cells DH5α. Selected clones were sequenced and verified to obtain a plasmid containing the mutation site S623N. Then use this plasmid as a template, carry out PCR amplification with synthetic amplification primers SEQ5 and SEQ6, utilize Dpn I enzyme to digest the original master plate after amplification, and the product of PCR amplification, that is, the plasmid containing the enzyme gene after mutation Transformed into competent cel...
Embodiment 2
[0068] Example 2: Whole blood tolerance PCR direct amplification
[0069] Add freshly collected and different types of anticoagulated blood into the PCR reaction system, and perform PCR amplification reaction on different types and different contents of anticoagulated blood with the purified MutTaq DNA polymerase. The exemplary reaction system is as follows:
[0070] 30mM Tris-HCl, 3mM MgCl 2 , 0.25% Brij-58, 65mM (NH4) 2 SO 4 , pH8.5, 0.5μM Primer, 0.2mM dNTP, 0.1U / μL Mut Taq, and different concentrations and different types of anticoagulated blood are directly used as templates for the reaction.
[0071] An exemplary reaction procedure is as follows:
[0072]
[0073] from figure 1 It can be seen that Mut Taq DNA polymerase can amplify 5%, 10%, 20%, 30% Na-EDTA anticoagulant, K-EDTA and Na-Heparin very well.
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