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LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection specific primers and liquid chip

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of easy sample contamination, high false positive rate, complicated operation, etc., and achieve the effect of consistent detection effect, good detection specificity and low cross-reaction rate

Inactive Publication Date: 2013-10-30
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional solid-phase chips have disadvantages such as high price, poor repeatability, and complicated operation, while other PCR-based detection technologies, such as direct sequencing and fluorescent quantitative PCR technology, have the disadvantages of low sensitivity, easy sample contamination, and high false positive rate. Disadvantages, again, both methods 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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  • LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection specific primers and liquid chip
  • LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection specific primers and liquid chip
  • LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection specific primers and liquid chip

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Experimental program
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Effect test

Embodiment 1L

[0033] Embodiment 1 LIG3 gene mutation detection liquid chip mainly includes:

[0034] 1. ASPE Primers

[0035] Specific primer sequences were designed for the wild-type and mutant types of the five common genotypes C142T, C214T, A179G, C158T and G80A of the LIG3 gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0036] Table 1 ASPE primer sequence of LIG3 gene (tag sequence + specific primer sequence)

[0037]

[0038] 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.

[0039] 2. Microspheres coated with anti-tag seque...

Embodiment 2

[0051] Example 2 Using the LIG3 gene mutation detection liquid chip described in Example 1 to detect the sample, the formulations of the various solutions are 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 5 common genotypes C142T, C214T, A179G, C158T, G80A of LIG3 gene in one step, the product sizes are 335bp, 333bp, 319bp, 354bp, 343bp, respectively, The sequences (SEQ ID NO.31-40) are shown in Table 3 abo...

Embodiment 3

[0103] The liquid phase chip of embodiment 3 different ASPE primers detects the SNP site of LIG3 gene

[0104] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0105] Taking LIG3 gene C142T, C214T, A179G and C158T 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 C142T, C214T, A179G and C158T, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.10, and correspondingly, the anti-tag sequence coated on the microsphere 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.

[0106] Table 7 Design of liquid phase chip preparation

[0107]

[0108] ...

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Abstract

The invention discloses an LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection liquid chip and specific primers. The liquid chip mainly comprises each ASPE (allele specific primer extension) primer formed by tag sequences at 5' terminal and specific primer sequences aiming at target gene mutation sites at 3' terminal, microspheres coated by anti-tag sequences as well as amplification primers, wherein the specific primer sequences are SEQ ID NO.11 and SEQ ID NO.12 aiming at C142T sites, SEQ ID NO.13 and SEQ ID NO.14 aiming at C214T sites, SEQ ID NO.15 and SEQ ID NO.16 aiming at A179G sites, SEQ ID NO.17 and SEQ ID NO.18 aiming at C158T sites and / or SEQ ID NO.19 and SEQ ID NO.20 aiming at G80A sites. The liquid chip has the advantages that the coincidence rate of the detection results of the detection liquid chip provided by the invention and a sequencing method is as high as 100%; and parallel detection of wild types and mutant types 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 specific primer for detecting LIG3 gene mutation and a liquid phase chip. Background technique [0002] Eukaryotic DNA ligase (DNA ligase) plays an important role in DNA replication, recombination and repair by catalyzing ATP-dependent double-strand DNA nick ligation. DNA ligase-III (DNA Ligase III, LIG3) is a unique ligase that can be located in both the nucleus and mitochondria. LIG3 participates in base excision repair and other Single strand break repair. The gene encoding LIG3 is located on chromosome 17q11.2-q12. The catalytic activity of LIG3 is critical for the maintenance and survival of mitochondrial DNA. Mutations in LIG3 may cause some human syndromes associated with defects in mitochondrial DNA replication and / or repair. disease. At present, studies have shown that LIG3 gene mutation is related to the occurrence and develop...

Claims

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Application Information

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