TERT gene mutation detection specific primers and liquid chip

A detection solution, specific technology, applied in the field of molecular biology

Active Publication Date: 2013-12-18
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods of TERT gene mutation mainly include: microarray technology, fluorescent quantitative PCR technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology (MALDI-TOF-MS) , microarray technology has problems such as high cost, complexity, low detection sensitivity, poor repeatability, narrow analysis range, complex synthesis and immobilization of probes, etc. In addition, because the hybridization is located on the surface of the solid phase, there is a certain degree of steric hindrance.
Fluorescent quantitative PCR technology has the disadvantages of low sensitivity, easy sample contamination, and high false positive rate. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology is a soft ionization technology that has powerful and mature functions in the detection of proteins and other biological macromolecules. However, in the field of nucleic acid detection, due to the particularity of nucleic acid molecules, the detection is subject to certain restrictions.

Method used

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  • TERT gene mutation detection specific primers and liquid chip
  • TERT gene mutation detection specific primers and liquid chip
  • TERT gene mutation detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The TERT gene mutation detection liquid chip described in this embodiment mainly includes:

[0038] 1. ASPE Primers

[0039] Specific primer sequences were designed for wild-type and mutant types of seven common genotypes of TERT gene, T174G, C98T, G162A, G168A, C209T, G151T and A129T. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0040] The ASPE primer sequence (tag sequence+specific primer sequence) of table 1 TERT gene

[0041]

[0042]

[0043] 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 100pmol / mL stock solution with 10mmol / LTris Buffer.

[0044] 2. Microsp...

Embodiment 2

[0056] Example 2 Detection of samples using the TERT gene mutation detection liquid chip described in Example 1

[0057] The formula of described various solutions is as follows:

[0058] 50mM MES buffer (pH5.0) formula (250ml):

[0059]

[0060] 2×Tm hybridization buffer

[0061]

[0062] Store at 4°C after filtration.

[0063] ExoSAP-IT kit was purchased from US USB Company.

[0064] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0065] 1. Sample DNA extraction:

[0066] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0067] 2. PCR amplification of samples to be tested

[0068] Design seven pairs of primers and multiplex PCR to amplify seven target sequences containing seven common genotypes of TERT gene T174G, C98T, G162A, G168A, C209T, G151T and A129T in one step, and the product sizes are 387bp, 220bp, 321bp, 292bp respectively , 334bp, 296bp, 297bp,...

Embodiment 3

[0113] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of TERT gene SNP site

[0114] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0115] Taking the liquid-phase chip for detection of site mutations of TERT genes T174G, C98T, G168A and G151T as an example, the specific primer sequences of the 3' end of the ASPE primers were designed for the wild-type and mutant types of T174G, C98T, G168A and G151T, respectively, and the ASPE primer 5 The Tag sequence at the '5 end is selected from SEQ ID NO.1-SEQ ID NO.14. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.29-SEQ ID NO.42. The specific design is shown in the following table (Table 8). 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. ...

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PUM

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Abstract

The invention discloses a TERT gene mutation detection liquid chip and specific primers. The liquid chip mainly comprises the following parts: each ASPE primer contains a tag sequence at the 5' terminal and a specific primer sequence aiming at target gene mutation locus at the 3' terminal, and the specific primer sequences are as below: a SEQ ID NO.15 and a SEQ ID NO.16 aiming at a T174G locus, a SEQ ID NO.17 and a SEQ ID NO.18 aiming at a C98T locus, a SEQ ID NO.19 and a SEQ ID NO.20 aiming at a G162A locus, a SEQ ID NO.21 and a SEQ ID NO.22 aiming at a G168A locus, a SEQ ID NO.23 and a SEQ ID NO.24 aiming at a C209T locus, a SEQ ID NO.25 and a SEQ ID NO.26 aiming at a G151T locus, and / or a SEQ ID NO.27 and a SEQ ID NO.28 aiming at an A129T locus; microballoons coated with anti-tag sequences; and amplification primers. The detection results from the detection liquid chip provided by the invention are 100% identical with those from a sequencing method, so as to realize parallel detection on wild types and mutants of a plurality of mutation loci.

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 TERT gene mutation detection and a liquid phase chip. Background technique [0002] TERT is called telomerase reverse transcriptase (Telomerase reverse transcriptase), which is located on chromosome 5 5P13.3. The TERT gene is a ribonucleoprotein located at the end of telomeres. Both are required for replication and for suppressing cellular senescence, and dysfunctional telomeres can affect gene stability and thereby increase the risk of tumor formation. The TERT gene encodes the catalytic subunit of telomerase, which is involved in the infinite proliferation of human cells and the pathogenesis of cancer cells. It is also a key gene in the development of cancer and is related to the occurrence of many cancers. It is related to basal cell carcinoma, lung cancer, bladder cancer, and prostate cancer. Cancer, cervical cance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
Inventor 许嘉森杨惠夷
Owner SUREXAM BIO TECH
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