ATR (ataxia telangiectasia-mutated and Rad3-related) gene mutation detection specific primers and liquid chip

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems that gene mutation detection cannot be used, can not meet the practical application, and the false positive rate is high, so as to avoid uncertain factors, the detection effect is consistent, and the detection specificity is good. Effect

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

[0003] At present, the detection methods of ATR gene mutation mainly include: PCR-RFLP, direct sequencing method and fluorescent quantitative PCR technology. PCR-RFLP method is based on the change of restriction enzyme recognition site caused by gene mutation, such as site loss or generation New site, a specific fragment is amplified by PCR, and then the amplified product is digested with restriction endonuclease, and the size of the fragment is observed by electrophoresis. Genotype, but ...

Method used

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  • ATR (ataxia telangiectasia-mutated and Rad3-related) gene mutation detection specific primers and liquid chip
  • ATR (ataxia telangiectasia-mutated and Rad3-related) gene mutation detection specific primers and liquid chip
  • ATR (ataxia telangiectasia-mutated and Rad3-related) gene mutation detection specific primers and liquid chip

Examples

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Effect test

Embodiment 1

[0020] Embodiment 1 ATR gene mutation detection liquid chip mainly includes:

[0021] 1. ASPE Primers

[0022] Specific primer sequences were designed for wild-type and mutant types of three common genotypes of ATR gene, G632A, T7301G and G946A. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0023] The ASPE primer sequence (tag sequence+specific primer sequence) of table 1ATR gene

[0024]

[0025] 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 / LTrisBuffer.

[0026] 2. Microspheres coated with anti-tag sequences

[0027] According t...

Embodiment 3

[0089] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of ATR gene mutation site

[0090] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0091] Taking the ATR gene G632A, T7301G and G946A 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 G632A, T7301G and G946A, and the tag sequence of the 5' end of the ASPE primer was It is selected from SEQ ID NO.1-SEQ ID NO.6, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. 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.

[0092] Table 7 Design of liquid...

Embodiment 4

[0105] The selection of embodiment 4ATR gene mutation detection specific primer sequence

[0106] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0107] Taking the liquid-phase chip for detecting the mutation site of ATR gene G632A and T7301G as an example, using the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template, design ASPE primers for the wild type and mutant type of G632A and T7301G, respectively The specific primer sequences at the 3' end include the preferred specific primer sequences and 2 alternative specific primer sequences in Example 1 of the present invention, as shown in Table 11. in, Inner bases are mutation sites.

[0108] Table 11 specific primer sequence

[0109]

[0110] Taking the mutation site detection liquid chip of ATR gene G632A and T7301G as an example, different specific primer sequences were selected for G632A and T7301...

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Abstract

The invention discloses an ATR (ataxia telangiectasia-mutated and Rad3-related) 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.7 and SEQ ID NO.8 aiming at G632A sites, SEQ ID NO.9 and SEQ ID NO.10 aiming at T7301G sites and/or SEQ ID NO.11 and SEQ ID NO.12 aiming at G946A 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 ATR gene mutation detection and a liquid phase chip. Background technique [0002] The ATR gene is located on chromosome 3 (3q22-q24), responsible for encoding ataxia telangiectasia and related protein Rad3 (Ataxiatelangiectasia-mutatedandRad3-related, ATR), ATR protein belongs to phosphatidylinositol 3-kinase-related kinase (phosphatidylinositol3 -kinaserelatedkinase, PIKK) protein family member, is the main member of the DNA damage checkpoint, they are activated by different types of DNA damage, by phosphorylating the corresponding downstream proteins Chk1 and Chk2, etc., regulate each checkpoint of the cell cycle, causing the cell cycle Therefore, ATR plays a crucial role in maintaining the stability of the genome. At present, studies have shown that ATR gene mutations are related to the occurrence and development o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C40B40/06
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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