Specific detection primers and detection liquid phase chip for chromosome Xp11.22 segment polymorphism

A polymorphism detection and liquid phase chip technology, applied in the field of molecular biology, can solve the problems of detection limitations, high false positive rate, complicated operation, etc., and achieve consistent detection results, good detection specificity, and low cross-reaction rate Effect

Active Publication Date: 2014-02-12
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a soft ionization technology that has powerful and mature functions in the detection of protein and other biological macromolecules. However, in the field of nucleic acid detection, due to the particularity of nucleic acid molecules, detection is subject to certain limit
Fluorescent quantitative PCR technology has the characteristics of high sensitivity, strong specificity, and high degree of automation, but it also has the disadvantages of easy sample contamination and high false positive rate, and can only detect one mutation type at a time
Although the Illumina fiber optic bead chip technology is a high-throughput detection system with high sensitivity and accuracy, it has a low degree of automation and many manual operations, which cannot meet the needs of practical applications.
However, the SNPlexTM System technology has high requirements for SNP sequence specificity, and cannot randomly analyze the selected single nucleotide polymorphism sites, so it is difficult to apply to clinical detection and diagnosis, and the method is complicated to operate and cannot meet the needs of practical applications.

Method used

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  • Specific detection primers and detection liquid phase chip for chromosome Xp11.22 segment polymorphism
  • Specific detection primers and detection liquid phase chip for chromosome Xp11.22 segment polymorphism
  • Specific detection primers and detection liquid phase chip for chromosome Xp11.22 segment polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 The liquid chip for detecting mutations in the chromosome Xp11.22 segment mainly includes:

[0042] 1. ASPE Primers

[0043] Specific primer sequences were designed for the wild-type and mutant types of three common genotypes A65G, G121A and C162T in the chromosome Xp11.22 segment. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0044] Table 1 ASPE primer sequence (tag sequence + specific primer sequence) of chromosome XP11.22 segment

[0045]

[0046] Each ASPE primer consists of 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 Table 1 above). 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.

[00...

Embodiment 2

[0058] Example 2 Using the chromosome Xp11.22 segment mutation detection liquid chip described in Example 1 to detect samples, the formulations of the various solutions are as follows:

[0059] 50mM MES buffer (pH5.0) formula (250ml):

[0060]

[0061] 2×Tm hybridization buffer

[0062]

[0063]

[0064] Store at 4°C after filtration.

[0065] ExoSAP-IT kit was purchased from US USB Company.

[0066] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0067] 1. Sample DNA extraction:

[0068] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0069] 2. PCR amplification of samples to be tested

[0070] Three pairs of primers were designed, and multiplex PCR amplified three target sequences containing three common genotypes A65G, G121A and C162T respectively in the chromosome Xp11.22 segment. NO.19-24) see Table 3 above.

[0071] First prepare the multiplex P...

Embodiment 3

[0112] Example 3 Detection of the SNP site of chromosome Xp11.22 segment by liquid chip with different ASPE primers

[0113] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0114] Taking the liquid-phase chip for detection of site mutations in the A65G, G121A and C162T segments of chromosome Xp11.22 as an example, the specific primer sequences of the 3' ends of the ASPE primers were designed for the wild-type and mutant types of A65G, G121A and C162T, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.6. 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 an...

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Abstract

The present invention discloses a detection liquid phase chip and specific primers for chromosome Xp11.22 segment polymorphism. The liquid phase chip mainly comprises: ASPE primers comprising a 5' terminal tag sequence and a 3' terminal target gene mutational site-targeted specific primer sequence, wherein the specific primer sequence comprises A65G site-targeted SEQ ID NO.7, A65G site-targeted SEQ ID NO.8, G121A site-targeted SEQ ID NO.9, G121A site-targeted SEQ ID NO.10, and / or C162T site-targeted SEQ ID NO.11 and C162T site-targeted SEQ ID NO.12; anti-tag sequence coated microspheres; and amplification primers. According to the present invention, coincidence frequency of the detection results of the detection liquid phase chip and the sequencing method is up to 100%, and single and parallel detection on the wild-type with multiple mutational sites and the mutant-type with multiple mutational sites can be 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 chromosome Xp11.22 segment polymorphism and a liquid phase chip. Background technique [0002] The chromosome Xp11.22 region is a gene-rich region for several neurodevelopmental disorders, and the identity of rare interstitial microdeletions of chromosome Xp11.22, craniofacial abnormalities and / or cleft lip / palate, and The truncating mutation of the PHF8 gene, the gene of language and cognitive ability in the region of chromosome Xp11. A large number of deletions in the .22 region, including the FAM120C and WNK3 genes, may be associated with the pathogenesis of autism. In addition, genetic mutations in the region of chromosome Xp11.22 are associated with the occurrence of prostate cancer. [0003] At present, the detection methods of chromosome Xp11.22 segment mutation mainly include: matrix-assisted laser d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q2600/156
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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