Single tube in situ nested polymerase chain reaction method and its use

A technology of chain reaction and polymerase, which is applied in the field of molecular biology, can solve problems such as cumbersome steps, easy pollution, and mutual influence of inner and outer primer amplification, and achieve the effect of simple operation, simple detection method, and reduced false negative results

Inactive Publication Date: 2006-11-08
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a single-tube in-situ nested PCR method to overcome the shortcomings of various existing nested PCR methods, such as complicated steps, easy pollution, mutual influence of inner and outer primer amplification, or the need for special reaction containers

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 The target gene is the human beta-actomyosin gene (No. BC016045 in the Gene Bank of the National Center for Biological Information, USA). The sequences and properties of the primers are shown in Tables 1 and 2, and the underlined sequences are non-gene-specific.

[0025] name

[0026] Primer name

[0027] When using cDNA as a template, the amplified products of the outer and inner sleeves are 939 and 401 bases in length, and the melting temperatures of the amplified products are 92.2 and 91.0°C, respectively. less than 2. The Advantage2-PCR kit of ClonTech Company was used, the primer was 0.4uM, and the reaction volume of each tube was 10ul. The nucleic acid template is cDNA, which is synthesized by reverse transcription from human tissue total RNA using ClonTech's reverse transcription kit with Oligo(dT) as a primer, and each tube contains 1ul of the cDNA prepared above. Table 3 shows the maximum allowable annealing temperatures for A ou...

Embodiment 2

[0034] Example 2 Design a nested PCR with a pair of outer primers and two sets of inner primers. The target gene, reagents and PCR procedure are the same as in Example 1. In addition to the outer and inner primers shown in Table 1, a pair of inner primers were added to the reaction solution, the sequence and characteristics of which are shown in Tables 5 and 6, and the underlined is the non-gene-specific sequence. The length of the amplified product of inner set primer 2 is 276 bases, and the melting temperature of the amplified product is 90.2°C

[0035] name

[0036]

[0037] Two bands with expected lengths of 401 bases and 279 bases could be obtained in PCR amplification.

Embodiment 3

[0039] Example 3 Design two pairs of outer primers and two pairs of inner primers for multiple nested PCR, the target gene, reagents and PCR program methods are the same as in Example 1. The sequences and properties of the two pairs of outer and two pairs of inner primers are shown in Tables 7 and 8, and the underlines are non-gene-specific sequences.

[0040] name

[0041]

[0042]PCR amplification can obtain two expected lengths of 197 bases and 275 bases.

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PUM

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Abstract

The present invention relates to single tube in-situ nested PCR method and its application in nucleotide detection. The reaction liquid contains both inside primer and outer primer, and the inner primer has mismatching with target gene sequence or one section of non-specific gene sequence in the 5' end. The inner primer and the original template have highest annealing temperature lower than that of the first round reaction, while the inner primer and the matched template have the melting temperature higher than the highest allowed annealing temperature of the outer primer. The first round PCR cycle annealing temperature is controlled for only the outer primer to be capable of annealing amplification, and the second round PCR cycle annealing temperature is raised for only the inner primer to be capable of annealing amplification. Between the first round and the second round, there are two or more transition stages with lower circular annealing temperature for the inner primer and the original template to form matching template to start the second round reaction.

Description

technical field [0001] The invention relates to molecular biology technology, in particular to a nested polymerase chain reaction method and its application. technical background [0002] Polymerase chain reaction (PCR) is a method for efficiently amplifying specific DNA in vitro. The nested PCR (Nested-PCR, Nested-PCR) developed on this basis has excellent amplification specificity and sensitivity. Significantly increased. However, the traditional nested PCR needs to open the cap of the reaction tube in two rounds, which is cumbersome and significantly increases the chance of lagging contamination. In the 1990s, a single-tube nested method based on controlling the concentration of low coat primers and the melting temperature of high coat primers was established, (Gookin et al: Journal-Clinical-Microbiology, 2002, 40, 4126, Forsman et al: Journal-Viological -Methods, 2003, 111: 1-11) This method requires experimentation to determine the appropriate coat primer concentratio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68
Inventor 徐定邦谢文凯府雷宇
Owner 徐定邦
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