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Specific primer and liquid phase chip for detecting TOX3 gene mutation

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of inappropriate clinical diagnostic chips, unable to meet actual needs, low degree of automation, etc., to avoid uncertain factors, consistent detection results, and simple steps. Effect

Active Publication Date: 2014-06-11
SUREXAM BIO TECH
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0003] At present, TOX3 gene mutation detection methods mainly include: Illumina fiber optic bead chip technology, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Affymetrix SNP 6.0 chip technology, although Illumina fiber optic bead chip technology is highly sensitive and accurate high-throughput detection system, but the degree of automation is low, and there are many manual operations, which are difficult to meet the needs of practical applications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology is a soft ionization technology. It has powerful and mature functions in detection, but in the field of nucleic acid detection, due to the particularity of nucleic acid molecules, detection is subject to certain restrictions
Although the high throughput makes the Affymetrix SNP 6.0 chip technology relatively mature, it is not suitable for low-to-medium density clinical diagnostic chips, and it is difficult to expand the detection of SNPs or tagged SNPs related to many biological traits in the same reaction system , In addition, the Affymetrix SNP 6.0 chip is mainly strong on the expression profile chip, with many species, relatively weak on the SNP chip, and the detection price is expensive, which cannot meet the actual needs

Method used

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  • Specific primer and liquid phase chip for detecting TOX3 gene mutation
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  • Specific primer and liquid phase chip for detecting TOX3 gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 TOX3 gene mutation detection liquid chip mainly includes:

[0031] 1. ASPE Primers

[0032] Specific primer sequences were designed for wild type and mutant types of five common genotypes T116C, A90C, G160A, C131T and G110A of TOX3 gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0033] Table 1 ASPE primer sequence of TOX3 gene (tag sequence + specific primer sequence)

[0034]

[0035]

[0036] 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 mutant or wild-type specific primer fragment (as shown in Table 1 above). 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.

[0037] 2. Microspheres coated with anti-tag sequences...

Embodiment 2

[0050] Example 2 Using the TOX3 gene mutation detection liquid chip described in Example 1 to detect samples

[0051] The formula of described various solutions is as follows:

[0052] 50mM MES buffer (pH5.0) formula (250ml):

[0053]

[0054] 2×Tm hybridization buffer

[0055]

[0056] Store at 4°C after filtration.

[0057] ExoSAP-IT kit was purchased from US USB Company.

[0058] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0059] 1. Sample DNA extraction:

[0060] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0061] 2. PCR amplification of samples to be tested

[0062] Design 5 pairs of primers, multiplex PCR to amplify 5 target sequences containing five common genotypes T116C, A90C, G160A, C131T and G110A of TOX3 gene in one step, the product sizes are 306bp, 168bp, 268bp, 321bp, 303bp, respectively, The sequences (SEQ ID NO.31-40) are shown in...

Embodiment 3

[0105] Example 3 Detection of TOX3 gene SNP site by liquid chip with different ASPE primers

[0106] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0107] Taking TOX3 gene T116C, A90C, C131T and G110A 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 T116C, A90C, C131T and G110A, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.10. Correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.21-SEQ ID NO.30. 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.

[0108] Table 7 Design of liquid phase chip ...

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Abstract

The invention discloses a liquid phase chip and a specific primer for detecting TOX3 gene mutation. The liquid phase chip mainly comprises: ASPE primers, microspheres coated by different anti-tag sequences, and an amplification primer, wherein each ASPE primer is composed of a tag sequence at a 5' end and specific primer sequences at a 3' end and aiming at target gene mutation sites, and the specific primer sequences are as follows: SEQ ID NO.11 and SEQ ID NO.12 aiming at a T116C site, SEQ ID NO.13 and SEQ ID NO.14 aiming at an A90C site, SEQ ID NO.15 and SEQ ID NO.16 aiming at a G160A site, SEQ ID NO.17 and SEQ ID NO.18 aiming at a C131T site, and / or SEQ ID NO.19 and SEQ ID NO.20 aiming at a G110A site. The coincidence rate of the detection result of the detection liquid phase chip disclosed by the invention with that of a sequencing method reaches as high as 100%, and parallel detection of a wild type and a mutant 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 TOX3 gene mutation detection specific primer and a liquid phase chip. Background technique [0002] The English name of the TOX3 gene is TOX high mobility group box family member 3, also known as trinucleotide repeat containing 9 (TNRC9) or CAGF9, located on chromosome 16 16q12.1. TOX3 gene encodes highly mobile group box protein, which is involved in the regulation of calcium ion-dependent transcription, which may be related to the bending and unwinding of DNA and the change of chromosome structure. Increased expression of the TOX3 gene can predict breast cancer metastases to the bone. New research identifies TOX3 gene as associated with genetic susceptibility to breast cancer. SNPs in TOX3 significantly increase the risk of breast cancer in women with BRCA mutations. The risk of breast cancer, if the gene FGFR2 exists in the body at the...

Claims

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Application Information

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IPC IPC(8): C40B40/06C12Q1/68C12N15/11
Inventor 刘丽胡文晖
Owner SUREXAM BIO TECH
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