Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene

A gene polymorphism and detection solution technology, applied in the field of molecular biology, can solve the problems of unsatisfactory practical application, poor timeliness, low sensitivity, etc., and achieve the effect of improving detection accuracy, consistent detection effect, and simple steps

Active Publication Date: 2014-04-16
广州益善医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, the methods for detection and analysis of DPYD gene polymorphism mainly include direct sequencing method and real-time fluorescent quantitative PCR technology. The sensitivity of direct sequencing method is only 20%-25%. The operation is complicated and the timeliness is poor, which cannot meet the needs of practical applications. Especially for heterogeneous tumor somatic mutations, low sensitivity will lead to a large number of missed detections; while real-time fluorescent quantitative PCR technology has a sensitivity of 1%-2%, high detection efficiency, and strong timeliness, but its high The false positive rate is also criticized for practical applications
Again, the above methods all have limitations in detection throughput, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.

Method used

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  • Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene
  • Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene
  • Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 DPYD gene polymorphism detection liquid chip mainly includes:

[0030] 1. ASPE Primers

[0031] Specific primer sequences were designed for wild-type and mutant types of three common genotypes of DPYD gene, G55A, G141A and G134T. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0032] Table 1 ASPE primer sequence of DPYD gene (tag sequence + specific primer sequence)

[0033]

[0034] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0035] 2. Microspheres coated with anti-tag sequences

[0036]According...

Embodiment 2

[0048] Example 2 Detection of samples using the DPYD gene polymorphism detection liquid chip described in Example 1

[0049] The formula of described various solutions is as follows:

[0050] 50mM MES buffer (pH5.0) formula (250ml):

[0051]

[0052] 2×Tm hybridization buffer

[0053] Reagent

[0054] Store at 4°C after filtration.

[0055] ExoSAP-IT kit was purchased from US USB Company.

[0056] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0057] 1. Sample DNA extraction:

[0058] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0059] 2. PCR amplification of samples to be tested

[0060] Three pairs of primers were designed, and multiplex PCR amplified three target sequences containing three common genotypes G55A, G141A, and G134T of the DPYD gene in one step. The product sizes were 231bp, 192bp, and 134bp. ) are shown in Table 3 above.

[0...

Embodiment 3

[0102] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of DPYD gene SNP site

[0103] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0104] Taking the liquid phase chip for detecting mutations at the G55A site of the DPYD gene as an example, the specific primer sequences at the 3' end of the ASPE primer were designed for the wild type and mutant type of G55A, and the tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.6. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0105] Table 7 Design of liquid phase chip preparatio...

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Abstract

The invention discloses a specific primer and liquid chip for detecting polymorphism of a DPYD (dihydropyrimidine dehydrogenase) gene. The liquid chip mainly comprises ASPE (allele specific primer extension) primers, microspheres and amplification primers, wherein each ASPE primer is formed by tag sequences at the 5' terminal and specific primer sequences which are arranged at the 3' terminal and aim at the mutation site of the target gene; the specific primer sequences are SEQ ID NO.7 and SEQ ID NO.8 aiming at the G55A site, SEQ ID NO.9 and SEQ ID NO.10 aiming at the G141A site and / or SEQ ID NO.11 and SEQ ID NO.12 aiming at the G134T site; and the microspheres are enveloped by anti-tag sequences. The detecting liquid chip provided by the invention has the advantages that the consistency of the detection result and the sequencing method is as high as 100%, thus parallel detection of wild types and mutant types of a plurality of mutation sites is achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a DPYD gene polymorphism detection specific primer and a liquid phase chip. Background technique [0002] Fluorouracil (5-Fu) is one of the most widely used antineoplastic drugs in the treatment of various tumors. More than 80% of 5-Fu in the body is catabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. The dihydropyrimidine dehydrogenase (DPD enzyme) encoded by the dihydropyrimidine dehydrogenase gene (DPYD gene) is the main rate-limiting enzyme in the metabolism of fluorinated pyrimidine antineoplastic drugs, and its activity has significant individual differences, and thus affects The curative effect and toxic and side effects of drugs, some studies have shown that the polymorphism of DPYD gene is significantly related to the enzyme activity encoded by it. [0003] The DPYD gene mutation site of target detection of the pres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 许嘉森吴诗扬
Owner 广州益善医学检验所有限公司
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