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

An activity detection and polymerase technology, applied in biochemical equipment and methods, microbial determination/inspection, fluorescence/phosphorescence, etc., can solve the problems of poor PCR product specificity, lack, detection of TaqDNA polymerase cold start activity, etc.

Inactive Publication Date: 2013-09-18
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 method
  • Taq DNA (deoxyribonucleic acid) polymerase cold start activity detection method
  • Taq DNA (deoxyribonucleic acid) polymerase cold start activity detection method

Examples

Experimental program
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Effect test

Embodiment 1

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

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

[0034] Refer to the full gene sequence of plasmid pGEM-5Zf(+) registered on GenBank, use PP5.0 software to design a pair of primers, the sequence is as follows: T1: 5'-GTTTTTCCATAGGCTCCGC-3', T2: 5'-TAGCACCGCCTACATACCTC-3', proceed Artificially synthesized, this pair of primers theoretically amplifies the sequence length of 401bp, and its base sequence is as SEQ ID NO:4. At the same time, design a fake template T3, the sequence is as follows: T3: 5'-AAGCAGTTCACTAGAGGTATGTAGGCGGTGCTA -3', where the 3'end of T3 is completely complementary to T2, and the 13 bases at the 5'end are the same as the template plasmid pGEM-5Zf(+) The sequence is completely different.

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

[0036] Dilute the sy...

Embodiment 2

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

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

[0050] Refer to the human HLA-DRA1 full gene sequence registered on GenBank, use the primer design software primer5.0 to design a pair of primers, the sequence is as follows: T4: 5'-ATCCCTACTCGCCATCATTC-3', T5: 5'-TCTCATCACCATCAAAGTCAAAC-3', artificially Synthesized, this pair of primers theoretically amplifies the sequence length of 223bp, and its base sequence is as SEQ ID NO: 8; at the same time, a fake template T6 is designed, the sequence is as follows: T6: 5'-TACTAGCGCGTG GTTTGACTTTGATGGTGATGAGA-3' where 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, which is artificially synthesized;

[0051] (...

Embodiment 3

[0064] The DNA polymerase cold-start activity test of the Taq DNA polymerase to be tested with the whole mouse gene as the template, the specific operation steps are as follows:

[0065] (1) Design and processing of primer and false template sequence

[0066] Refer to the full gene sequence of mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) registered on GenBank, use primer design software primer5.0 to design a pair of primers, the sequence is as follows: T7: 5'- CCCCTGTTTCTTGTCTTTCA-3', T8: 5'- GCTTCCCATTCTCGGCCTTG-3', artificially synthesized, this pair of primers theoretically amplify the sequence length of 191bp, and its base sequence is as SEQ ID NO: 12. Simultaneously design a fake template T9, the sequence is as follows: T9: 5'- CGAATGTGAC CAAGGCCGAGAATGGGAAGC-3', where the 3'end of T9 is completely complementary to T8, and the 10 bases at the 5'end are completely the same as the template mouse genomic DNA sequence Different, artificial synthesis;

[0067] (2) Treatme...

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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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Patent Type & Authority Patents(China)
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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