A kind of recombinant dna polymerase and its preparation method and application
A polymerase and recombinant carrier technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems of slow synthesis speed, unsatisfactory tolerance to interfering substances, poor affinity, amplification fragment length and amplification efficiency, etc., to achieve high selectivity , fast speed, good tolerance effect
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Embodiment 1
[0016] The present embodiment provides the preparation method of different mutant recombinant DNA polymerases as follows:
[0017] (1) Synthesize nucleotide sequences encoding DNA polymerases at different mutation sites. The synthesized nucleotide sequences include Nde I and Hind III enzyme cleavage sites as the target gene. Ligate the vector pET-30a(+) double-digested with Nde I and Hind III to the target gene with T4 DNA ligase at a reaction temperature of 16°C overnight to obtain recombinant vectors with different mutation sites.
[0018] (2) Mix the recombinant vector with Escherichia coli BL21 (DE3) competent cells, ice-bath for 30 minutes, heat shock at 42°C for 60 seconds, ice-bath for 5 minutes, add 500 μl of liquid LB medium, incubate at 37°C for 45 minutes, coat Spread on a plate containing kanamycin and culture overnight at 37°C. Escherichia coli BL21 (DE3) was selected as the host cell mainly because it can efficiently express the gene of the vector containing the...
Embodiment 2
[0031] This example provides a test for the amplification efficiency of different mutant recombinant DNA polymerases.
[0032] The experimental group is wild-type DNA polymerase with the amino acid sequence shown in SEQ ID NO.3. The DNA polymerase amino acid sequence has a total of 660 amino acids, of which the 1st to 591st positions are derived from DNA polymerase I (DNA Pol I, GenBank: KP993175.1). After multiple active sites and key functional areas of the DNA polymerase are predicted by bioinformatics technology, single point mutations are performed to prepare different mutant recombinant DNA polymerases. Single point mutations include: amino acid 249 from L to V (L249V), amino acid 269 from Y to P (Y269P), amino acid 299 from Q to K (Q299K), amino acid 313 from D Mutation to V (D313V), amino acid 314 from T to S (T314S), amino acid 315 from K to L (K315L), amino acid 335 from E to D (E335D), amino acid 359 Mutation from E to P (E359P), mutation of amino acid 453 from R ...
Embodiment 3
[0057] Embodiment 3 Recombinant DNA polymerase temperature gradient test
[0058] The wild-type DNA polymerase amino acid sequence has a total of 660 amino acids, of which the 1st to 591st positions are derived from DNA polymerase I. In this example, the D313V and K315L recombinant DNA polymerase linkage affinity sequences provided in Example 2 are used as the new recombinant DNA polymerase (Mut-D), wherein the 592nd to 597th are Linker (GTGGGG), and the 598th to 597th are Linker (GTGGGG). Position 660 is the affinity sequence VTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK.
[0059] The new recombinant DNA polymerase (Mut-D) was used as the experimental group, and the control group included the polymerase with D313V single-point mutation and no affinity sequence (Mut-313), and the polymerase with K315L single-point mutation without affinity sequence (Mut-D). -315), D313V and K315L double-site mutant polymerase without linking affinity sequence (Mut-313 / 315), ...
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