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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 the problems that cannot meet the practical application and cannot be used, and achieve the effect of improving detection accuracy, consistent detection effect, and avoiding uncertain factors

Active Publication Date: 2015-02-04
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 phase chip described in this embodiment mainly includes:

[0025] 1. ASPE primers

[0026] For HRAS gene Codon 12 normal genotype and five mutant types: G12S, G12C, G12R, G12D, G12V, Codon 13 normal genotype and three mutant types: G13S, G13R, G13C, and Codon 61 normal genotype And four mutants: Q61K, Q61R, Q61L, Q61H, design specific primer sequences respectively. ASPE primers consist of "tag sequence + specific primer sequence". The 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 consists of 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 primer fragment specific for mutant or wild type (as shown in Table 1 above). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service ...

Embodiment 2

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

[0044] The formulations of the various solutions are as follows:

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

[0046]

[0047] 2 x Tm hybridization buffer

[0048] reagent

[0049] After filtration, store at 4°C.

[0050] The ExoSAP-IT kit was purchased from the US USB company.

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

[0052] 1. DNA extraction of samples:

[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 containing 12 common genotypes of 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 p...

Embodiment 3

[0091] Example 3 Detection of HRAS gene mutation sites by liquid-phase chips with different ASPE primers

[0092] 1. Design of liquid 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 sequences at the 3' end of the ASPE primers were designed for the wild-type and mutant types of G12S and G13S respectively, and the Tag sequence at the 5' end of the ASPE primers was selected from SEQ ID NO: 1. ID NO.1-SEQ ID NO.15, correspondingly, the anti-tag sequences that are coated on the microspheres and paired with the corresponding tag sequences are 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, the coating of microspheres with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0094] Table 7 Design of liquid ...

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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, and in particular relates to a specific primer and a liquid phase chip for HRAS gene mutation detection. Background technique [0002] The ras gene is involved in the regulation of cell growth and differentiation, and is involved in the formation and development of various tumors. There are three characteristic genes associated with human tumors in the ras gene family, namely H-ras, K-ras and N-ras, which are located on chromosomes 11, 12, and 1, respectively. Activation of ras family oncogenes is the molecular basis for the occurrence and development of some tumors. Among them, point mutation is an important way of ras family gene activation, and the point mutation of the 12th, 13th and 61st codons of the ras family gene can make it acquired. ability to transform cells. Therefore, the detection of ras gene point mutations in tumor cells is of great signif...

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 SUREXAM BIO TECH