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ABO gene mutation detection specific primers and liquid chip

A technology of ABO gene and detection solution, which is applied in the field of molecular biology, can solve the problems of unsuitable gene mutation detection, unsuitable for practical application, and high false positive rate, so as to avoid uncertain factors, consistent detection effect and good detection specificity Effect

Inactive Publication Date: 2014-11-26
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, ABO gene mutation detection methods 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 this method cannot be used for the detection of gene mutations that do not generate new restriction sites
However, other PCR-based detection technologies, such as direct sequencing and fluorescent quantitative PCR technology, have the disadvantages of low sensitivity, easy contamination of samples, and high false positive rate.
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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  • ABO gene mutation detection specific primers and liquid chip
  • ABO gene mutation detection specific primers and liquid chip
  • ABO gene mutation detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0034] 1. ASPE Primers

[0035] Specific primer sequences were designed for wild type and mutant types of six common genotypes of ABO gene T98C, G268A, C124A, G100T, G139T and G50A. 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 ABO gene (tag sequence + specific primer sequence)

[0037]

[0038]

[0039] 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.

[0040] 2. Microspheres coated with anti-tag seq...

Embodiment 3

[0108] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of ABO gene SNP site

[0109] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0110] Taking the ABO gene T98C, G268A, G100T and G50A 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 T98C, G268A, G100T and G50A, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.12. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36. 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.

[0111] Table 8 Design of l...

Embodiment 4

[0127] Embodiment 4 ABO gene mutation detection specific primer sequence selection

[0128] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0129] Taking the liquid-phase chip for detecting mutation sites of ABO genes C124A and G139T as an example, use the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template to design ASPE primers for the wild type and mutant types of C124A and G139T, 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 12. in, Inner bases are mutation sites.

[0130] Table 13 specific primer sequence

[0131]

[0132]

[0133] Taking the mutation site detection liquid chip of ABO gene C124A and G139T as an example, different specific primer sequences were selected for C124A and G1...

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Abstract

The invention discloses an ABO 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.13 and SEQ ID NO.14 aiming at T98C sites, SEQ ID NO.15 and SEQ ID NO.16 aiming at G268A sites, SEQ ID NO.17 and SEQ ID NO.18 aiming at C124A sites, SEQ ID NO.19 and SEQ ID NO.20 aiming at G100T sites, SEQ ID NO.21 and SEQ ID NO.22 aiming at G139T sites and / or SEQ ID NO.23 and SEQ ID NO.24 aiming at G50A 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 ABO gene mutation and a liquid phase chip. Background technique [0002] The human ABO gene is located on chromosome 9q34.1-34.2, and its gene product is glycosyltransferase, which controls the biosynthesis of ABO blood group antigen. The ABO gene contains 7 exons ranging in length from 28 to 688 and 6 introns with a length of about 19514 bp, and the total length is about 18 to 20 kb. Most of the coding sequences are located on exons 6 and 7, which encode the catalytic domain of the glycosyltransferase. At present, studies have shown that ABO gene mutations are associated with the occurrence and development of chronic pancreatitis and pancreatic cancer, plasma levels, and intercellular adhesion molecule levels, and have a great relationship with the risk of pancreatic cancer. [0003] At present, ABO gene mu...

Claims

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Application Information

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