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Factor II and Factor V genetic polymorphism detection specific primer and liquid-phase chip

A gene polymorphism and detection solution technology, which is applied in the fields of medicine and biology, can solve problems such as being unusable and unable to meet the needs of practical applications, and achieve consistent detection results, simple steps, and good detection specificity

Active Publication Date: 2014-08-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
In addition, 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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  • Factor II and Factor V genetic polymorphism detection specific primer and liquid-phase chip
  • Factor II and Factor V genetic polymorphism detection specific primer and liquid-phase chip
  • Factor II and Factor V genetic polymorphism detection specific primer and liquid-phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Factor II and Factor V gene polymorphism detection liquid chip mainly includes:

[0025] 1. ASPE Primers

[0026] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes A140G, G78A and G125A of Factor II and Factor V genes, respectively. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0027] Table 3 ASPE primer sequences of Factor II and Factor V genes (Tag sequence + specific primer sequence)

[0028]

[0029]

[0030] 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 mm...

Embodiment 2

[0042] Example 2 Using the Factor II and Factor V gene polymorphism detection liquid chip described in Example 1 to detect samples The formulations of the various solutions are as follows:

[0043] 50mM MES buffer (pH5.0) formula (250ml):

[0044]

[0045]

[0046] 2×Tm hybridization buffer

[0047] Reagent source Final concentration Dosage per 250ml 1M Tris-HCl, pH8.0 SigmaT3038 0.2M 50ml 5M NaCl Sigma S5150 0.4M 20ml Triton X-100 Sigma T8787 0.16％ 0.4ml

[0048] Store at 4°C after filtration.

[0049] ExoSAP-IT kit was purchased from US USB Company.

[0050] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0051] 1. Sample DNA extraction:

[0052] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0053] 2. PCR amplification of samples to be tested

[0054] Three pairs of primers were designed, and ...

Embodiment 3

[0097] Example 3 Detection of Factor II and Factor V gene polymorphism sites by liquid chip with different ASPE primers

[0098] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0099]Taking the liquid-phase chip for detection of site mutation of Factor II gene A140G and Factor V gene G125A as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of A140G and G125A, and the tag sequence of the 5' end of the ASPE primer was It is selected from SEQ ID NO.1-SEQ ID NO.6, and 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 9). 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.

[0100]...

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Abstract

The invention discloses a Factor II and Factor V genetic polymorphism detection specific primer and liquid-phase chip. The liquid-phase chip mainly comprises an ASPE (Allele Specific Primer Extension) primer, microspheres and an amplification primer, wherein the ASPE primer consists of a tag sequence at 5' end and specific primers aiming at target gene mutation at 3' end, wherein the specific primer sequences are SEQ ID NO. 7 and SEQ ID NO. 8 aiming at an A140G site of Factor II gene, SEQ ID NO. 9 and SEQ ID NO. 10 aiming at G78A site of Factor II gene, and / or SEQ ID NO. 11 and SEQ ID NO. 12 aiming at G125A of Factor V gene; and the microspheres are coated by anti-tag sequence. The matching ratio of the detection result of the Factor II and Factor V genetic polymorphism detection liquid-phase chip provided by the invention and that of a sequencing method reaches up to 100 percent. Parallel detection of wild type and mutant type of a plurality of polymorphic sites can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for detecting Factor II and Factor V gene polymorphisms and a liquid phase chip. Background technique [0002] The gene polymorphisms of prothrombin factor II (Factor II) and coagulation factor V (Factor V) are research hotspots. At present, there are few reports on the detection and analysis of Factor II and Factor V gene polymorphisms, and the research methods are mainly direct sequencing and PCR-RFLP analysis. The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. The size of the fragment is observed by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH