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PCR method of multiple primer, its reaction liquor and application for preparing detection reagent

A reaction solution and multiplex technology, applied in the field of molecular biology, to achieve the effect of increasing specificity and sensitivity and eliminating complexity

Inactive Publication Date: 2004-10-13
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to provide a polymerase chain reaction method with multiple primers and its reaction solution and its application in the preparation of detection reagents, so as to break through the limitations of multiple primer design in the existing multiple PCR method and overcome the limitations of multiple primers in a single tube. The defect that a PCR reaction cannot independently and simultaneously amplify multiple fragments of the same or different DNA sequences

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Comparison of ultra-low denaturing temperature multiplex PCR with conventional multiplex PCR.

[0028] Three pairs of primers were designed on the human actomyosin gene (GenBank No. BC016045), and their sequences and properties are listed in Tables 2 and 3, respectively.

[0029] name

[0030] Primer

[0031] Since the three pairs of genes target the same target gene and are relatively close to each other, unpaired amplification products will be formed under conventional multiplex PCR conditions, and their lengths and melting temperatures are shown in Table 4.

[0032] unpaired primers

[0033] The PCR reaction of Example 1 adopts the Advantage-2 kit of Clontech, the reaction volume of each tube is 5 microliters, the final concentration of each primer is 0.1uM, and each reaction tube contains 1 microliter of cDNA, and the cDNA is prepared from human tissue total RNA with Poly (dT)18 is a primer synthesized according to its regulations usi...

Embodiment 2

[0038] Multiplex PCR with 6 pairs of primers.

[0039] The primer design is shown in Table 6.

[0040] The primer properties are listed in Table 7.

[0041] The characteristics of the amplification products with unpaired primers are shown in Table 8.

[0042] name

[0043] Primer

[0044] The base length of human actomyosin is less than 2000 bases, and 6 pairs of primers have the possibility of 15 kinds of unpaired combination amplification. The measured length and melting temperature of the amplified products are shown in Table 8.

[0045]

[0046] The melting temperatures of the six paired amplification products were all lower than 76.8°C, while the melting temperatures of the 15 unpaired amplification products were all higher than 80.9°C. Example 2 The reaction reagents and conditions are the same as in Example 1, but the subsequent cycle of denaturation is 79°C for 30 seconds. As a result, any combination of A04-A09 primer pairs includin...

Embodiment 3

[0048] Design scheme and implementation conditions of ultra-low denaturation temperature multiplex PCR with 10 pairs of primers.

[0049] The Cyclin-D gene (GenBank No. NC_053056) was analyzed with the primer design software Oligo, and 10 pairs of primers with high stringency and the melting temperature of the amplified products were all lower than 81°C were searched. The positions and characteristics of 10 pairs of primers and the characteristics of 45 unpaired interference amplification products are listed in Table 9 and Table 10.

[0050] Primer pair number

[0051] Primer pair number

[0052] The results show that the Tm value of the interference product is between 82.1-92.1°C, and most of them are between 83-87°C, which is obviously not in the same temperature range as the normal product, that is to say, it is possible to obtain in the 4300-base sequence of the gene 10 pairs of primers meet the design requirement that the melting temperature of the a...

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Abstract

The present invention relates to a PCR method of multiple primer, its reaction liquid and its application in preparation of microbial detection reagent, specially relates to that in PCR process two denaturation temp. of 92-97 deg.C and 65-87 deg.C are adopted to respectively make initial 2 or 3 circulation or other subsequent circulation so as to overcome the defect of existent multiple PCR which can form non-specific product. Said invention utilizes the proper product design and adoption to two denaturation temperatures so as to obviously reduce the possibility of that non-target product early-synthesized by PCR can participate in subsequence. Said invention is applicable to preparation of single-tubule multiple PCR quick detection reagent, and has extensive application for detecting various viruses.

Description

technical field [0001] The invention relates to molecular biology technology, in particular to a polymerase chain reaction method, its reaction solution and its application in the preparation of detection reagents. technical background [0002] The classic polymerase chain reaction method was invented in the mid-1980s. It uses a forward primer and a reverse primer to amplify a specific gene segment. Soon, a multiplex PCR (Multiplex-PCR) method was developed in which two or more pairs of primers were used to simultaneously amplify several products in one reaction tube. Several primer pairs of current multiplex PCR are usually complementary to several different target gene sequences, which are carried on independent cDNAs, or carried on the same genomic DNA but separated from each other by a considerable distance. The current multiplex PCR is often used to compare the expression levels of different genes. In clinical testing, multiplex PCR can be used to detect two or more pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/04C12Q1/68
Inventor 徐定邦朱德芬陈有容徐文慧
Owner 徐定邦
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