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VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip

A detection solution and specific technology, applied in the field of molecular biology, can solve problems such as inability to meet practical applications and cannot be used, and achieve the effects of improving detection accuracy, consistent detection effect, and low cross-reaction rate.

Active Publication Date: 2013-04-10
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.
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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  • VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip
  • VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip
  • VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1 VHL gene mutation detection liquid chip mainly includes:

[0021] 1. ASPE Primers

[0022] Specific primer sequences were designed for wild type and mutant types of six common genotypes of VHL gene T240A, T254C, T266A, C286T, G388C and 444delT. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0023] The ASPE primer sequence (tag sequence+specific primer sequence) of table 1 VHL gene

[0024]

[0025]

[0026] 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.

[0027] 2. Microspheres coated with anti-t...

Embodiment 2

[0038] Example 2 Using the VHL gene mutation detection liquid chip described in Example 1 to detect samples

[0039] The formula of described various solutions is as follows:

[0040] 50mM MES buffer (pH5.0) formula (250ml):

[0041]

[0042] 2×Tm hybridization buffer

[0043] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5MNaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16%

0.4ml

[0044] Store at 4°C after filtration.

[0045] ExoSAP-IT kit was purchased from US USB Company.

[0046] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0047] 1. Sample DNA extraction:

[0048] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0049] 2. PCR amplification of samples to be tested

[0050] Design 2 pairs of p...

Embodiment 3

[0099] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of VHL gene mutation site

[0100] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0101] Taking the VHL gene T240A and T254C site mutation detection liquid chip 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 T240A and T254C, and the tag sequence of the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.12, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36. 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.

[0102] Table 7 Design of liquid phase chip preparation ...

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PUM

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Abstract

The invention discloses a VHL (Von Hippel Lindau) genetic mutation detection specific primer and a liquid phase chip. The liquid phase chip mainly comprises ASPE (Allele Specific Primer Extension) primers, microspheres coated by an anti-tag sequence, and amplimers, wherein the ASPE primers consist of tag sequences at a 5' end and specific primer sequences specific to target genetic mutation sites at a 3' end; and the specific primer sequences are: SEQ ID NO.13 and SEQ ID NO.14 specific to a T240A site; SEQ ID NO.15 and SEQ ID NO.16 specific to a T254C site; SEQ ID NO.17 and SEQ ID NO.18 specific to a T266A site; SEQ ID NO.19 and SEQ ID NO.20 specific to a T286T site; SEQ ID NO.21 and SEQ ID NO.22 specific to a G388C site; and / or SEQ ID NO.23 and SEQ ID NO.24 specific to a 444delT site. The matching rate between a detection result of the liquid phase chip provided by the invention and a sequencing method is up to 100 percent, and wild and mutant parallel detection of a plurality of mutant sites is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a VHL gene mutation detection specific primer and a liquid phase chip. Background technique [0002] VHL gene is named after Von Hippel-Lindau (VHL) disease, contains 14543bp, includes 3 exons and 2 introns. VHL disease is a clinically very rare familial autosomal dominant genetic tumor disease, including central nervous system hemangioblastoma, visceral tumor and cyst, etc. In 1993, Latif et al. located the VHL gene on chromosome 3p25-26 through linkage analysis , and successfully cloned the VHL gene for the first time. The VHL gene is a tumor suppressor gene. The inactivation of this gene will lead to the disorder of normal VHL protein synthesis, which is closely related to the occurrence and development of clear cell renal cell carcinoma (CCRCC). At present, important progress has been made in the research on the structure and function ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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