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Specific detection primers and detection liquid phase chip for FYCO1 gene mutation

A technology of specificity and detection solution, which is applied in the field of molecular biology, can solve the problems of time-consuming and labor-intensive sequencing reactions, high false positive rate, and insufficient flexibility, so as to avoid uncertain factors, good detection specificity, and avoid cross-reactions Effect

Inactive 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

[0003] At present, the FYCO1 gene mutation detection methods mainly include: ABI 3730xl conventional sequencing platform and fluorescent quantitative PCR technology. ABI 3730xl sequencing technology can achieve high-throughput detection, but it is not flexible enough. It can only perform qualitative analysis on the detection results, and it is difficult to operate. It is very cumbersome, and the sequencing reaction is time-consuming and laborious. 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

Method used

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  • Specific detection primers and detection liquid phase chip for FYCO1 gene mutation
  • Specific detection primers and detection liquid phase chip for FYCO1 gene mutation
  • Specific detection primers and detection liquid phase chip for FYCO1 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 FYCO1 gene mutation detection liquid chip mainly includes:

[0027] 1. ASPE Primers

[0028] Specific primer sequences were designed for the wild-type and mutant types of two common genotypes A92G and C57T of the FYCO1 gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0029] Table 1 ASPE primer sequence (specific primer sequence) of FYCO1 gene

[0030]

[0031]

[0032] Table 2 ASPE primer sequence (tag sequence) of FYCO1 gene

[0033]

[0034] Each ASPE primer includes two parts, the 5' end is the specific tag sequence for the anti-tag sequence on the corresponding microsphere (as shown in the above table 2), and the 3' end is the 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 100 pmol / mL stock solut...

Embodiment 2

[0048] Example 2 Detection of samples using the FYCO1 gene mutation detection liquid chip described in Example 1

[0049] The formula of described various solutions is as follows:

[0050] 50mM MES buffer (pH5.0) formula (250ml):

[0051]

[0052] 2×Tm hybridization buffer

[0053] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0054] Store at 4°C after filtration.

[0055] ExoSAP-IT kit was purchased from US USB Company.

[0056] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0057] 1. Sample DNA extraction:

[0058] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0059] 2. PCR amplification of samples to be tested

[0060] Design 2 pair...

Embodiment 3

[0104] The liquid phase chip of embodiment 3 different ASPE primers detects the SNP site of FYCO1 gene

[0105] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0106] Taking the FYCO1 gene A92G and C57T site mutation detection liquid chip as an example, the specific primer sequence at the 3' end of the ASPE primer was designed for the wild type and mutant type of A92G and C57T, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.5-SEQ ID NO.10, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.11-SEQ ID NO.16. 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.

[0107] Table 9 Design of liquid phase chip preparation

[0108]

...

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Abstract

The present invention discloses a detection liquid phase chip and specific primers for FYCO1 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 A92G site-targeted SEQ ID NO.1, A92G site-targeted SEQ ID NO.2, and / or C57T site-targeted SEQ ID NO.3 and C57T site-targeted SEQ ID NO.4; 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 FYCO1 gene mutation and a liquid phase chip. Background technique [0002] The FYCO1 gene is called FYVE and coiled-coil domain containing 1, and its English name is FYVE and coiled-coil domain containing 1. It is located on the short arm of chromosome 3 3p21.31, specifically between 45959394 and 46037306 base pairs on chromosome 3. FYCO1 gene mutation is the cause of an autosomal recessive hereditary cataract, and it is believed to play a role in the biological variation of the lens and the pathogenesis of autosomal recessive congenital cataract. Studies have found that the protein synthesized by this gene has autophagy, which is a necessary process for eliminating harmful proteins. To keep the lens healthy, the cells on it must constantly break down certain protein substances. The researchers believe that a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6883C12Q2600/156
Inventor 许嘉森何嘉英
Owner SUREXAM BIO TECH
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