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Specific detection primers and detection liquid phase chip for KLK3 gene mutation

A detection solution and specificity technology, applied in the field of molecular biology, can solve problems such as difficult clinical detection and diagnosis, easy contamination of samples, complicated method operation, etc., and achieve the effects of avoiding uncertain factors, consistent detection results, and avoiding cross-reactions

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

[0003] At present, the KLK3 gene mutation detection methods mainly include: Illumina fiber optic bead chip technology, SNPlex Genotyping System and fluorescence quantitative PCR technology, although Illumina fiber optic bead chip technology is a high-throughput detection system with high sensitivity and accuracy, but the degree of automation is low , there are many manual operations, which are difficult to meet the needs of practical applications. The SNPlexTM System technology has high requirements for SNP sequence specificity, and the selected SNPs cannot be randomly typed, so it is difficult to apply to clinical detection and diagnosis. Meet the needs of practical applications
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, which also cannot meet the needs of practical applications.

Method used

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  • Specific detection primers and detection liquid phase chip for KLK3 gene mutation
  • Specific detection primers and detection liquid phase chip for KLK3 gene mutation
  • Specific detection primers and detection liquid phase chip for KLK3 gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1 KLK3 gene mutation detection liquid chip mainly includes:

[0021] 3. ASPE Primers

[0022] Specific primer sequences were designed for wild-type and mutant types of five common genotypes of KLK3 gene, G11453A, G7670A, A9367C, T536C and A15G. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0023] Table 1 ASPE primer sequence of KLK3 gene (tag sequence + specific primer sequence)

[0024]

[0025]

[0026] 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.

[0027] 2. Microspheres coated with anti-tag s...

Embodiment 2

[0040] Example 2 Detection of samples using the KLK3 gene mutation detection liquid chip described in Example 1

[0041] The formula of described various solutions is as follows:

[0042] 50mM MES buffer (pH5.0) formula (250ml):

[0043]

[0044] 2×Tm hybridization buffer

[0045]

[0046] Store at 4°C after filtration.

[0047] ExoSAP-IT kit was purchased from US USB Company.

[0048] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0049] 1. Sample DNA extraction:

[0050] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0051] 2. PCR amplification of samples to be tested

[0052] Design 5 pairs of primers, multiplex PCR to amplify 5 target sequences containing five common genotypes of KLK3 gene G11453A, G7670A, A9367C, T536C and A15G respectively, the product sizes are 330bp, 296bp, 251bp, 445bp, 294bp The sequences (SEQ ID NO.31-40) are shown in Table ...

Embodiment 3

[0095] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of KLK3 gene SNP site

[0096] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0097] Taking the KLK3 gene G11453A, G7670A, T536C and A15G 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 G11453A, G7670A, T536C and A15G, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.10. Correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence 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.

[0098] Table 7 De...

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Abstract

The present invention discloses a detection liquid phase chip and specific primers for KLK3 gene mutation. 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 G11453A site-targeted SEQ ID NO.11, G11453A site-targeted SEQ ID NO.12, G7670A site-targeted SEQ ID NO.13, G7670A site-targeted SEQ ID NO.14, A9367C site-targeted SEQ ID NO.15, A9367C site-targeted SEQ ID NO.16, T536C site-targeted SEQ ID NO.17, T536C site-targeted SEQ ID NO.18, and / or A15G site-targeted SEQ ID NO.19 and A15G site-targeted SEQ ID NO.20; 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 KLK3 gene mutation detection specific primer and a liquid phase chip. Background technique [0002] Kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen (PSA), KLK3 gene is located at 19q13.2-q13.4, the full length of the gene is 6KB, containing 5 There are 4 exons and 4 introns, the full length of the transcribed mRNA is 1446bp, and the transcription is regulated by androgen. The KLK3 gene is a member of the human tissue kallikrein gene (KLK) family, and is a tumor marker widely used clinically for screening and diagnosing prostate cancer. PSA has been found to be expressed in a variety of tissues and body fluids, with the most abundant concentration in seminal plasma. Because PSA has serine protease and chymotrypsin-like activity, it can specifically degrade serine protease-like and chymotrypsin-specific subst...

Claims

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Application Information

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