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Method for detecting target nucleotide sequence using competitive primer

A competitive primer and base sequence technology, which is used in the detection of target base sequences using competitive primers, can solve problems such as low extension efficiency, double-stranded instability, and reduced extension efficiency.

Inactive Publication Date: 2015-11-25
TOPPAN PRINTING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0025] The methods proposed in Patent Documents 2 to 3 are methods of introducing the same variation at the same position between competing primers, and this method utilizes the property that if the primer matches the target nucleic acid near the 3' end, it can effectively The extension reaction is carried out efficiently, the more the number of mismatched bases, the lower the extension efficiency
In addition, with the increase of mismatched bases, the duplex between the primer and the target nucleic acid becomes unstable as a whole, which is also related to the decrease of the extension efficiency

Method used

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  • Method for detecting target nucleotide sequence using competitive primer
  • Method for detecting target nucleotide sequence using competitive primer
  • Method for detecting target nucleotide sequence using competitive primer

Examples

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Embodiment

[0242] In Comparative Examples 1 to 2 and Examples 1 to 2, the gene at position 1173 in the intron 1 region of vitamin K epoxide reductase complex 1 (VKORC1), which is a gene related to the optimal dose of warfarin, was more The state (1173C>T) is set as the recognition object.

[0243] As shown in Table 9, two detection primers (VK1Wat-Acridine and VK1Mtg-Acridine) and a common primer (VK1R2) for detecting the above-mentioned gene polymorphism of VKORC1 were produced. As primers for detection, acridine was introduced at the 5' end using acridine phosphoramide (manufactured by Glenresearch), and 6-fluorescein (manufactured by Glenresearch) was introduced into its 5' end purchased from Japan BioServices Co. 'Terminal detection primers. For the four kinds of competitive primers (VK1Mtg, VK1Mat, VK1Mac, VK1Wat) and common primer (VK1R2) shown in Table 9, primers synthesized by a usual synthesis method were purchased from Greiner Bio-One. As the genomic DNA as a template, a gene...

Embodiment 1）

[0260] The same reaction and the same analysis as in Comparative Example 1 were performed except that VK1Wat-Acridine was used as the detection primer and VK1Mtg was used as the competitive primer. express the result in Figure 13 middle.

[0261] Figure 13 It is a graph showing the result of Example 1 as a negative first-order differential curve of the melting curve.

[0262] When the combination of the detection primer and the competitive primer of Example 1 was used, the amplification of VK1Wat-Acridine was preferentially observed when the C allele was used as a template. In addition, compared with the results of Comparative Example 2, the amplification of VK1Wat-Acridine was more suppressed when the T allele was used as a template, and it was found that the C allele was more specific in the above primer set. Excellent.

Embodiment 2）

[0264] The same reaction and the same analysis as in Comparative Example 1 were performed except that VK1Mtg-Acridine was used as the detection primer and VK1Wat was used as the competitive primer. express the result in Figure 14 middle.

[0265] Figure 14 It is a graph showing the result of Example 2 as a negative first-order differential curve of the melting curve.

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Abstract

Disclosed is a method of detecting a target base sequence having a polymorphic base, the method including: (a) a step of adding to a nucleic acid sample having a target nucleic acid that includes a base sequence including the target base sequence: at least one type of detection primer, at least one type of competitive primer, and at least one type of common primer; (b) a step of annealing the detection primer and the competitive primer to the target nucleic acid in a competitive manner, thereby synthesizing an extension product A; (c) a step of annealing the common primer to the extension product A obtained in the step (b) or in the following step (d), thereby synthesizing an extension product B; (d) a step of annealing the detection primer or the competitive primer to the extension product B obtained in the previous step (c), thereby synthesizing the extension product A; and (e) a step of detecting the extension product A or the extension product B.

Description

technical field [0001] The present invention relates to a method for detecting a target base sequence having a polymorphic base and a kit for use in the method. More specifically, it relates to a detection method for detecting a target nucleotide sequence with high accuracy by competition of primers, and a kit used for the method. [0002] The present application claims priority based on the invention patent application No. 2010-68490 filed with the Japan Patent Office on March 24, 2010, the content of which is incorporated herein by reference. Background technique [0003] In recent years, according to the global analysis of the human genome, 3.1 billion base pairs of sequences have been determined, and the number of human genes has been determined to be about 30,000 to 40,000. [0004] There are different base sequences between different individuals of human beings. If the base sequence exists at a frequency of more than 1% in a specific population, it is called a genetic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/6869C12Q1/6827C12Q1/6858C12Q2531/119C12Q2537/161C12Q2531/143C12Q2531/113C12Q2525/161C12Q2525/185C12Q2525/204C12Q2535/125C12Q2549/101C12Q2563/107C12Q2565/101
Inventor 牧野洋一山根明男中山雅人
Owner TOPPAN PRINTING CO LTD
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