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Taq DNA polymerase mutant, PCR reaction reagent and kit

A polymerase and mutant technology, applied in the direction of enzymes, applications, transferases, etc., can solve problems such as confusion, increased reagent costs and time costs, and cross-contaminated samples

Pending Publication Date: 2021-03-30
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, further studies have found that the above nucleic acid combinations and kits can be further optimized from the following aspects: (1) The test sample is a human DNA sample extracted from EDTA anticoagulated whole blood, and a commercial kit is required to extract human genomic DNA before testing, not only The cost of reagents and time is increased, and there is a risk of cross-contamination or sample confusion due to improper operation during the extraction process. Therefore, it is necessary to further optimize the PCR reaction solution to achieve Y without extracting genomic DNA and directly using blood samples as templates. The purpose of accurate detection of chromosomal microdeletion; (2) the DNA polymerase in its Taqman probe multiplex fluorescent PCR reaction system is the DNA polymerase of routine transformation, in the Taqman probe method fluorescent PCR reaction, Taq DNA polymerase needs to have at the same time Very good polymerase activity and 5'-3'exonuclease activity to ensure amplification efficiency and the efficiency of fluorescent signal release, while conventional Taq DNA polymerase modification and modification programs focus on improving polymerase performance while ignoring external Therefore, it is necessary to further optimize and modify DNA polymerase to achieve better detection results.

Method used

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  • Taq DNA polymerase mutant, PCR reaction reagent and kit
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  • Taq DNA polymerase mutant, PCR reaction reagent and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] This embodiment provides a Taq DNA polymerase mutant whose amino acid sequence is shown in SEQ ID NO.25.

[0072] The present embodiment also provides the preparation method of above-mentioned Taq DNA polymerase mutant, it comprises the following steps:

[0073] (1) Sequence optimization is carried out to synthesize the gene fragment of the Taq DNA polymerase mutant (identical to the nucleotide sequence shown in the 1-2496th position in SEQ ID NO.27), and the group is added at the 3' end of the gene fragment Amino acid tag (shown as positions 2695-2724 in SEQ ID NO.27).

[0074] (2) Ligate the nucleotide fragment obtained in step (1) into the gene expression vector pET-28a to construct a recombinant plasmid.

[0075] (3) Transforming the recombinant plasmid into the host cell, ie Escherichia coli competent cell E. coli BL21(DE3), to obtain the recombinant cell.

[0076] (4) Induce the expression of the recombinant cells in step (3), and collect the thalline; the speci...

Embodiment 2

[0090]This embodiment provides a Taq DNA polymerase mutant complex, which is formed by fusion of the Taq DNA polymerase mutant in Example 1 and the DNA binding protein of SEQ ID NO.26.

[0091] SEQ ID NO.26:

[0092] MVKVKFKYKGEEKEVDTSKIKKVWRVGKMVSFTYDDNGKTGRGAVSEKDAPKELLDMLARAEREKK.

[0093] The Taq DNA polymerase mutant complex adopts the nucleotide sequence shown in SEQ ID NO.27 (the underline is a histidine tag, and the nucleotide sequence encoding the DNA binding protein shown in SEQ ID NO.26 is the 2497-2694th position ), prepared with reference to the method of Example 1. Adjust the concentration of the complex to the appropriate concentration using the enzyme stock solution. Wherein, the enzyme storage solution contains: 20mM Tris-HCl (pH 8.0), 100mM KCl, 1mM dithiothreitol and 40% glycerol.

[0094] SEQ ID NO.27:

[0095] atgcgtggtatgttacgattatttgaaccaaaaggtcgtgttttattggttgatggtcatcatctggcctatcgtacctttcatgcactgaagggattgacaacctcccgtggcgaacgcgtccaggcagtgtatggattcgca...

Embodiment 3

[0097] This embodiment provides another Taq DNA polymerase mutant complex, which is formed by antigen-antibody reaction between the Taq DNA polymerase mutant in Example 1 and the nanobody against the Taq DNA polymerase mutant.

[0098] The preparation method of the above nanobody can refer to the following steps:

[0099] (a) Take an appropriate amount of the Taq DNA polymerase mutant obtained in Example 1 as an antigen, and add an adjuvant. In this example, Fisher's adjuvant is used to immunize alpacas that can produce nanobodies. Stop immunization when the antibody production is stable. Blood collection, using affinity chromatography or adsorbent method to separate the antiserum, after measuring the specificity and titer of the antiserum, separating and purifying the antiserum to obtain the anti-Taq DNA polymerase mutant nanobody;

[0100] (b) Preparation of antigen-antibody complex: the anti-Taq DNA polymerase mutant nanobody obtained in the step (a) of the anti-Taq DNA pol...

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Abstract

The invention discloses a Taq DNA polymerase mutant, a PCR reaction reagent and a kit, and relates to the technical field of gene detection. The amino acid sequence of the Taq DNA polymerase mutant disclosed by the invention is as shown in SEQ ID NO. 25. When the Taq DNA polymerase mutant is used for PCR reaction, a blood sample can be directly used as a template for detection, and the Taq DNA polymerase mutant has the characteristics of short detection time, high sensitivity, strong specificity, high accuracy and the like.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to Taq DNA polymerase mutants, PCR reaction reagents and kits. Background technique [0002] At present, infertility plagues about 10%-15% of couples of childbearing age in the world, of which male factors account for about 50% (Bushnik T et al., Hum Reprod, 2012, 27:738-746). There are many factors that cause male infertility. In addition to endocrine hormone disorders, reproductive tract inflammation, varicocele, immune abnormalities, and physical and chemical factors, about 30% of them are caused by sperm development disorders caused by genetic defects (Bhasin S et al. , J Clin Endocrinol Metab,1994,79:1525-1529; Chang P L et al., Human Reprod,1999,14:2689-2694), and Y chromosome microdeletion is one of the main genetic factors (Luddi A et al ., N Engl J Med, 2009, 360:881-885). [0003] Therefore, the detection of Y chromosome AZF microdeletion has important clinical si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12Q1/686
CPCC12N9/1252C12Q1/686C12Y207/07007
Inventor 吴诗扬许嘉森刘志明刘芳
Owner SUREXAM BIO TECH