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Taq DNA (deoxyribonucleic acid) polymerase cold start activity detection metho

A technology for activity detection and polymerase, which is applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc. It can solve the problems of poor specificity of PCR products, base mismatches, and high temperature resistance of Klenow fragments, etc.

Inactive Publication Date: 2012-07-04
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantages are: ① Klenow fragments are not resistant to high temperature, and will be denatured and inactivated at 90°C, so they must be added again every cycle
②The primer chain extension reaction is carried out at 37°C, which is prone to base mismatch between the template and the primer, the specificity of the PCR product is poor, and the synthesized DNA fragments are not uniform
We searched the literature and found that there is still a lack of objective and effective technical means to detect the cold-start activity of Taq DNA polymerase

Method used

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  • Taq DNA (deoxyribonucleic acid) polymerase cold start activity detection metho
  • Taq DNA (deoxyribonucleic acid) polymerase cold start activity detection metho
  • Taq DNA (deoxyribonucleic acid) polymerase cold start activity detection metho

Examples

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Effect test

Embodiment 1

[0032] Use the plasmid pGEM-5Zf(+) as a template to detect the DNA polymerase cold-start activity of the Taq DNA polymerase to be tested. The specific steps are as follows:

[0033] (1) Design and synthesis of primers and pseudo-templates

[0034] Referring to the complete gene sequence of the plasmid pGEM-5Zf (+) registered on GenBank, a pair of primers were designed using PP5.0 software. Artificially synthesized, the theoretically amplified sequence length of this pair of primers is 401bp, and its base sequence is as shown in SEQ ID NO:4. Simultaneously design the false template T3, the sequence is as follows: T3: 5'-AAGCAGTTCACTAGAGGTATGTAGGCGGTGCTA -3', wherein, the 3' end of T3 is completely complementary to T2, and the 13 bases at the 5' end are compatible with the template plasmid pGEM-5Zf (+) The sequence is completely different.

[0035] (2) Treatment of false templates and primers before PCR reaction

[0036] Dilute the synthesized primers T1 and T2 and the false ...

Embodiment 2

[0048] The DNA polymerase cold-start activity detection of the Tth DNA polymerase to be tested is performed using the whole human gene as a template, and the specific operation steps are as follows:

[0049] (1) Design and synthesis of primers and pseudo-templates

[0050] Referring to the complete human HLA-DRA1 gene sequence registered on GenBank, a pair of primers were designed using the primer design software primer5.0. Synthesized, the theoretically amplified sequence length of this pair of primers is 223bp, and its base sequence is shown in SEQ ID NO: 8; at the same time, a false template T6 is designed, and the sequence is as follows: T6: 5'-TACTAGCGCGTG GTTTGACTTTGATGGTGATGAGA-3' Among them, the 3' end of T6 It is completely complementary to T5, and the 12 bases at the 5' end are completely different from the corresponding sequence of the HLA-DRA1 gene of the template human genome, and are artificially synthesized;

[0051] (2) Treatment of false templates and primers...

Embodiment 3

[0064] The DNA polymerase cold-start activity detection of the Taq DNA polymerase to be tested is performed using the whole mouse gene as a template. The specific operation steps are as follows:

[0065] (1) Design and processing of primers and pseudo-template sequences

[0066] Referring to the complete gene sequence of mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) registered on GenBank, a pair of primers were designed using the primer design software primer5.0. The sequence is as follows: T7: 5'- CCCCTGTTTCTTGTCTTTCA-3', T8: 5'- GCTTCCCATTCTCGGCCTTG-3' is artificially synthesized, and the theoretically amplified sequence length of this pair of primers is 191bp, and its base sequence is as shown in SEQ ID NO:12. Simultaneously design a false template T9, the sequence is as follows: T9: 5'- CGAATGTGAC CAAGGCCGAGAATGGGAAGC-3', wherein, the 3' end of T9 is completely complementary to T8, and the 10 bases at the 5' end are completely complementary to the sequence of the ...

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Abstract

The invention relates to a Taq DNA (deoxyribonucleic acid) polymerase cold start activity detection method which comprises the following steps: designing primers and a false template according to a template, mixing the forward primer or reverse primer which is complementary withthe 5'terminal part of the false template, and carrying out denaturalizing annealing reaction to obtain the mixture;Adding the mixture and various constituents required by the PCR reaction, including template DNA, 5'terminal part, forward primer or reverse primer which is not complementary with the false template, PCR buffer, magnesium chloride, dNTP (deoxyribonucleotide triphosphate) mixture and Taq DNA polymerase to be measured, into a PCR tube to carry out PCR reaction; and judging whether the detected DNA polymerase has cold start activity according to the detection result of the PCR product. The method is simple to operate, has the characteristic of sensitivity, can effectively detect very weak cold start activity, and overcomes the defects of subjectivity and arbitrariness in the existing method.

Description

technical field [0001] The invention relates to a method for detecting enzyme activity, in particular to a method for detecting cold-start activity of Taq DNA polymerase. Background technique [0002] In 1985, Kary.B.Mullis of Cetus Company in the United States invented the epoch-making polymerase chain reaction (Polymerase Chain Reaction, PCR), which realized people's dream of infinitely amplifying DNA fragments in vitro. For the first time, Saiki successfully amplified human β-globin DNA by PCR method, and applied it to the prenatal diagnosis of sickle cell anemia. [0003] In the initial stage of the invention of PCR, Mullis used a very simple three-temperature water bath to conduct experiments, and used the Klenow fragment of Escherichia coli DNA polymerase Ⅰ to catalyze the extension effect of the annealing primer. Its disadvantages are: ① Klenow fragments are not resistant to high temperature, and will be denatured and inactivated at 90°C, so they must be added again ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48G01N21/64
Inventor 李智涛任爱红高秀菊张王丽王丽军冯艳铭
Owner HENAN UNIV OF SCI & TECH
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