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Specific detection primers and detection liquid phase chip for SLC22A3 gene mutation

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of high false positive rate, many manual operations, and low degree of automation, and achieve the effects of good detection specificity, avoiding cross-reaction, and low cross-reaction rate

Active Publication Date: 2014-02-12
SUREXAM BIO TECH
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  • Summary
  • 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
However, fluorescent quantitative PCR technology has the disadvantages of easy sample contamination, high false positive rate, and the disadvantage of only detecting 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.
Although the PCR-SSCP technology is simple to operate, there are false negatives and false positives. Generally, the detection rate of the fragments to be tested is not more than 300bp, and the detection rate is 70-80%. It can only detect whether there is a mutation, but cannot distinguish the specific Mutated base types, it is difficult to meet the needs of automation, and it is difficult to work on a large scale

Method used

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  • Specific detection primers and detection liquid phase chip for SLC22A3 gene mutation
  • Specific detection primers and detection liquid phase chip for SLC22A3 gene mutation
  • Specific detection primers and detection liquid phase chip for SLC22A3 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 SLC22A3 gene mutation detection liquid chip mainly includes:

[0035] 3. ASPE Primers

[0036] Specific primer sequences were designed for wild type and mutant types of six common genotypes of SLC22A3 gene, G161A, G191T, C150T, T182C, C91A and C144T. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0037] Table 1 ASPE primer sequence of SLC22A3 gene (tag sequence + specific primer sequence)

[0038]

[0039]

[0040] 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.

[0041] 2. Microspheres coated with an...

Embodiment 2

[0054] Example 2 Detection of samples using the SLC22A3 gene mutation detection liquid chip described in Example 1

[0055] The formula of described various solutions is as follows:

[0056] 50mM MES buffer (pH5.0) formula (250ml):

[0057]

[0058] 2×Tm hybridization buffer

[0059]

[0060] Store at 4°C after filtration.

[0061] ExoSAP-IT kit was purchased from US USB Company.

[0062] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0063] 1. Sample DNA extraction:

[0064] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0065] 2. PCR amplification of samples to be tested

[0066] Design 6 pairs of primers and multiplex PCR to amplify 6 target sequences containing six common genotypes G161A, G191T, C150T, T182C, C91A and C144T of the SLC22A3 gene in one step, and the product sizes are 361bp, 360bp, 330bp, 353bp, 234bp , 261bp, and the primer sequences (...

Embodiment 3

[0111] Example 3 Detection of the SNP site of SLC22A3 gene by liquid chip with different ASPE primers

[0112] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0113] Taking SLC22A3 gene G161A, G191T, T182C and C144T 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 5G161A, G191T, T182C and C144T, 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.

[0114] Table 8 Design of liq...

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Abstract

The present invention discloses a detection liquid phase chip and specific primers for SLC22A3 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 G161A site-targeted SEQ ID NO.13, G161A site-targeted SEQ ID NO.14, G191T site-targeted SEQ ID NO.15, G191T site-targeted SEQ ID NO.16, C150T site-targeted SEQ ID NO.17, C150T site-targeted SEQ ID NO.18, T182C site-targeted SEQ ID NO.19, T182C site-targeted SEQ ID NO.20, C91A site-targeted SEQ ID NO.21, C91A site-targeted SEQ ID NO.22, and / or C144T site-targeted SEQ ID NO.23 and C144T site-targeted SEQ ID NO.24; 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 SLC22A3 gene mutation and a liquid phase chip. Background technique [0002] The English name of the SLC22A3 gene is Solute carrier family 22 member 3, also known as organic cation carrier 3 (OCT3) or extraneuronal monoamine transmitter carrier (EMT), located on chromosome 6 6q25.3. The SLC22A3 gene encodes cation transporter 3 (humans encode EMT protein, rats encode OCT3 protein), and are widely distributed in tissues and organs such as prometaphase placenta, skeletal muscle, prostate, aorta, liver, salivary gland, and adrenal gland. SLC22A3 gene is a multispecific transmitter carrier independent of sodium ion. The transported substrates include histamine, serotonin, norepinephrine, dopamine, and 1-methyl-4-phenylpyridinium, and the ability to transport them and the affinity for these substrates differ betwe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6876C12Q2600/156
Inventor 许嘉森何嘉英
Owner SUREXAM BIO TECH
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