HRAS gene mutation detection specificity primer and liquid chip thereof

A detection solution and specificity technology, which is applied in the field of molecular biology, can solve problems such as unusable and unsatisfactory for practical applications, achieve consistent detection results, improve detection accuracy, and avoid uncertain factors

Active Publication Date: 2013-03-27
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.
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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  • HRAS gene mutation detection specificity primer and liquid chip thereof
  • HRAS gene mutation detection specificity primer and liquid chip thereof
  • HRAS gene mutation detection specificity primer and liquid chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The HRAS gene mutation detection liquid chip described in this embodiment mainly includes:

[0025] 1. ASPE Primers

[0026] For the normal genotype and five mutant types of HRAS gene Codon 12: G12S, G12C, G12R, G12D, G12V, the normal genotype and three mutant types of Codon 13: G13S, G13R, G13C, and the normal genotype of Codon 61 And four mutant types: Q61K, Q61R, Q61L, Q61H, respectively design specific primer sequences. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0027] Table 1 ASPE primer sequence of HRAS gene (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 Techno...

Embodiment 2

[0043] Example 2 Using the HRAS gene mutation detection liquid chip described in Example 1 to detect samples

[0044] The formula of described various solutions is as follows:

[0045] 50mM MES buffer (pH5.0) formula (250ml):

[0046]

[0047] 2×Tm hybridization buffer

[0048] Reagent

[0049] Store at 4°C after filtration.

[0050] ExoSAP-IT kit was purchased from US USB Company.

[0051] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0052] 1. Sample DNA extraction:

[0053] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0054] 2. PCR amplification of samples to be tested

[0055] Two pairs of primers were designed, and two target sequences of 12 common genotypes containing HRAS genes Codon12, Codon13, and Codon61 were amplified in one step by multiplex PCR. Among them, Codon12 and Codon13 were located in the same amplification product, and th...

Embodiment 3

[0091] Example 3 Detection of HRAS gene mutation site by liquid chip with different ASPE primers

[0092] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0093] Taking the HRAS gene G12S and G13S 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 G12S and G13S respectively, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.15, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.31-SEQ ID NO.45. 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.

[0094] Table 7 Design of liquid phase chip preparation

[0095] ...

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Abstract

The invention discloses an HRAS gene mutation detection specificity primer and a liquid chip thereof. The liquid chip mainly comprises: an ASPE primer composed of a 5'-terminal tag sequence and 3'-terminal specificity primer sequences focused on target gene mutation sites, wherein the specificity primer sequences comprise SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and / or SEQ ID NO.21 focused on a Codon12 site, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and / or SEQ ID NO.25 focused on a Codon13 site, and / or SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 and / or SEQ ID NO.30 focused on a Codon61 site; a microsphere coated by an anti-tag sequence; and an amplimer. The consistency between the detection result of the detection liquid chip provided by the invention and the detection result of a sequencing method is high to 100%, and the wild-type and mutant parallel detection of a plurality of mutation 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 HRAS gene mutation detection specific primer and a liquid phase chip. Background technique [0002] ras gene participates in the regulation of cell growth and differentiation, and participates in the formation and development of various tumors. There are three characteristic genes related to human tumors in the ras gene family, namely H-ras, K-ras and N-ras, which are located on chromosome 11, 12 and 1 respectively. The activation of ras family oncogenes is the molecular basis for the occurrence and development of some tumors. Among them, point mutations are an important way for the activation of ras family genes, and the point mutations of the 12th, 13th and 61st codons of ras family genes can make them acquire ability to transform cells. Therefore, the detection of ras gene point mutations in tumor cells is of great significance for un...

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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