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Polymorphic detection specific primers and liquid phase chip in 8 q 24 section of chromosome

A technology of polymorphism detection and liquid phase chip, which is applied in the field of molecular biology, can solve the problems of poor repeatability and unusability, and achieve good signal-to-noise ratio, consistent detection effect, and low cross-reaction rate

Inactive Publication Date: 2013-03-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
Thirdly, the traditional solid-phase chip cannot meet the needs of practical applications due to its poor repeatability, while other methods based on PCR technology 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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  • Polymorphic detection specific primers and liquid phase chip in 8 q 24 section of chromosome
  • Polymorphic detection specific primers and liquid phase chip in 8 q 24 section of chromosome
  • Polymorphic detection specific primers and liquid phase chip in 8 q 24 section of chromosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 The liquid-phase chip for detecting the polymorphism of chromosome 8q24 segment mainly includes:

[0022] 1. ASPE Primers

[0023]Specific primer sequences were designed for the wild-type and mutant types of 11 common genotypes G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T on chromosome 8q24 segment. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0024] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence) of chromosome 8q24 segment

[0025]

[0026]

[0027]

[0028] 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...

Embodiment 2

[0042] Example 2 Detection of Samples Using a Chromosome 8q24 Segment Detection Liquid Chip

[0043] The formula of described various solutions is as follows:

[0044] 50mM MES buffer (pH5.0) formula (250m1):

[0045]

[0046] 2×Tm hybridization buffer

[0047] Reagent

[0048] Store at 4°C after filtration.

[0049] ExoSAP-IT kit was purchased from US USB Company.

[0050] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0051] 1. Sample DNA extraction:

[0052] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0053] 2. PCR amplification of samples to be tested

[0054] 11 pairs of primers were designed, and multiplex PCR amplified 11 target sequences of 11 common genotypes G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T in one step, respectively containing the chromosome 8q24 segment , the product sizes are 273bp, 342b...

Embodiment 3

[0101] Example 3 Detection of Chromosome 8q24 Segment Polymorphism Site by Liquid Chip with Different ASPE Primers

[0102] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0103] Taking the liquid-phase chip for detection of site mutations in the C160T and C202A segments of chromosome 8q24 as an example, the specific primer sequences at the 3' end of the ASPE primers were designed for the wild-type and mutant types of C160T and C202A, respectively, and the Tag sequence at the 5' end of the ASPE primers was selected. From SEQ ID NO.1-SEQ ID NO.22, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.45-SEQ ID NO.66. The specific design is shown in the following table (Table 9). 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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Abstract

The invention discloses specific primers and liquid phase chip for polymorphic detection in an 8 q 24 section of a chromosome. The liquid phase chip comprises specific primers consisting of tag sequences at the 5' end and specific primer sequences aiming at polymorphic loci of a target gene at the 3' end, microspheres and an amplification primer, wherein the specific primer sequences are selected from more than one pair of SEQ ID No. 23 and SEQ ID No. 24 for G83T, SEQ ID No. 25 and SEQ ID No. 26 for C140A, SEQ ID No. 27 and SEQ ID No. 28 for C160T, SEQ ID No. 29 and SEQ ID No. 30 for G99A, SEQ ID No. 31 and SEQ ID No. 32 for C202A, SEQ ID No. 33 and SEQ ID No. 34 for C296T, SEQ ID No. 35 and SEQ ID No. 36 for A125C, SEQ ID No. 37 and SEQ ID No. 38 for T119C, SEQ ID No. 39 and SEQ ID No. 40 for C149A, SEQ ID No. 41 and SEQ ID No. 42 for C193T and SEQ ID No. 43 and SEQ ID No. 44 for G201T; and the tag sequences are selected from SEQ ID No. 1 to SEQ ID No. 22. The prepared liquid phase chip for the polymorphic detection in the 8 q 24 section of the chromosome have a high signal noise ratio, and cross reaction is not formed between a designed probe and anti-tag sequences basically.

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 chromosome 8q24 segment and a liquid phase chip. Background technique [0002] Genome-wide association studies have found that single nucleotide polymorphisms (single nucleotide polymorphisms, SNPs) on chromosome 8q24 are associated with genetic susceptibility to various tumors such as prostate cancer, colon cancer, and breast cancer. [0003] At present, there are many methods for detecting and analyzing the single nucleotide polymorphism of chromosome 8q24 segment, such as: direct sequencing method, PCR-RFLP analysis method, traditional solid-phase chip, etc., among which the most commonly used is PCR-RFLP Analysis. The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific frag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森郭婧罗小笛
Owner SUREXAM BIO TECH