PCR method with unique primer and its application

A technology of chain reaction and polymerase, applied in the field of molecular biology

Inactive Publication Date: 2004-11-24
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In conclusion, so far, no research report has tried and successfully applied the only primer to amplify a nucleic acid with a known sequence and obtain a specific target product

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The human actomyosin gene (1841 bases, GenBank number BC016045) is commonly used as a housekeeping gene and as a reference template in quantitative analysis and comparison of gene expression. In this example, the gene was tested as an example, and dozens of oligonucleotide sequences with reverse complementary lengths of 6-12 bases were found, some of which could meet the requirements of primers or high-stringency primers.

[0065] Table 5 and Table 6 list the sequence of primers found in the human actomyosin gene, which can be used as the only primer PCR method, and analyze the primers and the upstream and downstream binding sites of the template, the binding efficiency of the primers and the template, and other primers. characteristic.

[0066] Table 5. Sequences of some of the unique primers found in the human actomyosin gene

[0067] lead

[0068] * Bold type indicates a mismatch with the template order

[0069] Table 6. Properties ...

Embodiment 2

[0073] The same method as in Example 1 was used to design a unique primer for another housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase gene (1283 bases, NM_006959). Table 7 and Table 8 list the part of the primers found in this gene that can be used as the primer sequence of the unique primer PCR method, and analyze its primer and template upstream and downstream binding sites, primer and template binding efficiency and other characteristics of the primer.

[0074] Table 7. Sequences of some of the unique primers found in human glyceraldehyde-3-phosphate dehydrogenase

[0075] lead

[0076] * Bold type indicates a mismatch with the template order

[0077] Table 8. Properties of some of the unique primers found in human glyceraldehyde-3-phosphate dehydrogenase

[0078] lead

[0079] Table 8 shows that at least 5 oligonucleotides obtained in a gene with a length of about 1300 bases can be used for unique primer PCR, and their b...

Embodiment 3

[0081] Example 3 used the primers G2, G3 or G4 listed in Table 7 and Table 8 for amplifying the human glyceraldehyde-3-phosphate dehydrogenase gene. The volume of each tube of PCR reaction solution is 5 microliters, the primer concentration is 0.5 μM, and the complementary DNA is used as the template DNA. The complementary DNA is prepared from the total RNA of human tissues using the cDNA synthesis kit of ClonTech Company and using poly(dT) as the reverse transcription primer. get. The amount of human tissue total RNA used in reverse transcription is 1 microgram per 20 microliters of reaction solution. After the reverse transcription is completed, the reaction volume is diluted to 100 microliters with deionized water for use. Add 10 microliters of the above-mentioned diluted complementary DNA to every 100 microliters of PCR reaction solution. Advantage2 kit of ClonTech Company was used for PCR reaction. The reaction conditions were initial denaturation at 95°C for 1 minute, ...

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Abstract

The present invention provides the PCR or RT-PCR method with unique prime and its application, and the unique primer is oligomeric nucleotide chain designed based on the template DNA chain order characteristic and has 3' end with 6-12 bases in complementary order with the template and 5' end flexibly designed. The method is superior to classical PCR method, which has one pair of primers and maybe negative effect on proliferation efficiency and the complementing between 3' end bases in forward and inverse primers. The present invention is suitable for use in the PCR or RT-PCR test of human and pathological microbe gene, in multiple PCR or RT-PCR to reduce the mutual effect between primers and as the inner reference reaction of other PCR or RT-PCR, and is favorable to obtaining quantitative or semi-quantitative results.

Description

technical field [0001] The present invention relates to molecular biology techniques, in particular to methods involving polymerase chain reaction. technical background [0002] PCR is a simple, specific, sensitive and rapid gene amplification method. The basic principle and method of PCR is to design a pair of primers (primer-pair) according to the known gene sequence, wherein, the positive primer (forward-primer) is complementary to the anti-strand sequence in the template DNA double strand upstream of the template sequence, and The reverse primer (reverse-primer) is complementary to the positive strand sequence in the template DNA duplex downstream of the template sequence. The template DNA is denatured at high temperature and unraveled into a single strand. When the forward primer and reverse primer are lowered to the annealing temperature range, they are combined with the anti-strand and forward strand of the template respectively, and then DNA polymerase presses the 5...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68
Inventor 徐定邦朱德芬谢文凯徐文慧
Owner 徐定邦
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