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Specific primer for detecting fluorouracil medicament healing effect related gene mutation, liquid phase chip thereof and method thereof

A technology of fluorouracil and detection solution, which is applied in the field of molecular biology, can solve the problems of poor repeatability of detection results, expensive solid-phase chips, and high false positive rate, and achieve accurate and reliable detection results, improve detection accuracy, and simple steps Effect

Active Publication Date: 2010-01-13
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, several PCR-based technologies for detecting gene mutations have been established, such as direct sequencing, PCR-single-strand conformation mutation analysis (SSCP) detection, and the above technologies have disadvantages such as low sensitivity, easy contamination of samples, and high false positive rate.
However, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis technology and the allelic difference analysis method based on TaqMan technology can only detect one mutation at a time, which is time-consuming and laborious; traditional solid-phase chips Expensive, low sensitivity and poor reproducibility of test results

Method used

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  • Specific primer for detecting fluorouracil medicament healing effect related gene mutation, liquid phase chip thereof and method thereof
  • Specific primer for detecting fluorouracil medicament healing effect related gene mutation, liquid phase chip thereof and method thereof
  • Specific primer for detecting fluorouracil medicament healing effect related gene mutation, liquid phase chip thereof and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 The liquid-phase chip kit for detecting gene mutations related to the curative effect of fluorouracil drugs mainly includes:

[0033] 1. ASPE Primers

[0034] Specific primer sequences were designed for various common mutation site alleles of genes related to the efficacy of fluorouracil drugs. ASPE primers consist of Tag+ specific primer sequences. Specific primer sequences are shown in the table below:

[0035] Table 1 Specific primer sequences of ASPE primers

[0036]

[0037] Table 2 Tag sequences at the 5' end of ASPE primers

[0038]

[0039]

[0040] Each ASPE primer includes two parts, the 5' end is the tag sequence (as shown in Table 2) complementary to the anti-tag sequence (as shown in Table 3) on the corresponding microsphere, and the 3' end is for the mutant or wild Type-specific primer sequences (as shown in Table 1 above). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesi...

Embodiment 2

[0055] Example 2 Detection of Clinical Samples Using a Fluorouracil Drug Curative Effect-Related Gene Mutation Detection Liquid Chip The formulations of the various solutions are as follows:

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

[0057] Reagent

[0058] 2×Tm hybridization buffer

[0059] Reagent

[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] Follow the instructions of the AxyPrep Whole Blood Genome Mini Extraction Kit to obtain the DNA of the sample to be tested.

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

[0066] Six pairs of primers were designed using Primer5.0, and a total of 6 target sequences with mutation sites were amplified by multiplex PCR in one step. The product sizes were 592bp, 412bp, 708bp, 623bp, 534bp and 357bp. ...

Embodiment 3

[0119] Embodiment 3 Selection of Tag sequence and Anti-Tag sequence:

[0120] 1. Design of liquid phase chip preparation

[0121] Taking the detection liquid chip of the C667T site mutation of the MTHFR gene as an example, the specific primers at the 3' end of the ASPE primer are designed for the wild type and mutant type of C667T, and the Tag sequence at the 5' end of the ASPE primer is selected from SEQ ID NO.37 - 3 of SEQID NO.48, correspondingly, the anti-tag sequences coated on the microspheres and complementary to the corresponding tag sequences are selected from SEQ ID NO.13-SEQ ID NO.24. The specific design is shown in the following table (Table 5). The synthesis of ASPE primers, Anti-tag sequence coated microspheres, amplification primers, detection methods, etc. are as described in Example 1.

[0122] table 5

[0123]

[0124] 2. Sample testing

[0125] Using the liquid phase chip prepared by the above design, serum samples 1-10 were detected according to the ...

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Abstract

The invention discloses a specific primer for detecting fluorouracil medicament healing effect related gene mutation, a liquid phase chip thereof and a method thereof. The liquid phase chip comprises wild type primer pair and mutation specific ASPE primer pair which are respectively designed for each type of mutation position point, microballoon which respectively comprises specific anti-tag sequence, and a primer which is used for enlarging target sequence with DPYD, TS and MTHFR gene mutation position points. The liquid phase chip for detecting the fluorouracil medicament healing effect related gene mutation has extreme good signal-noise ratio, and cross reaction is basically unavailable between a designed probe and the anti-tag sequence. The designed ASPE primer has extreme good specificity and can exactly distinguish the each type of mutation position point. The detecting method has simple step, can detect six type mutation position points at one time, has convenient operation, and avoids various uncertain factors existed in many operation processes, thereby being capable of greatly heightening the detecting accuracy rate.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to specific primers for detecting gene mutations related to the curative effect of fluorouracil drugs, a liquid phase chip and a detection method thereof. Background technique [0002] 5-FU (fluorouracil, fluorouracil) was invented in 1957 by Heidelberger and Ansfield. 5-FU belongs to the fluorouracil class of antimetabolite drugs, such drugs include tegafur, carmofur, floxuridine, deoxyfluridine, and capecitabine. These drugs are converted into 5-FU in the body, and then play an anti-tumor effect. 5-FU was one of the first drugs to be used, and it is still an indispensable broad-spectrum antineoplastic drug in cancer treatment. It has a good curative effect on digestive system cancer (esophageal cancer, gastric cancer, intestinal cancer, pancreatic cancer, liver cancer) and breast cancer, and is also effective on cervical cancer, ovarian can...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森何嘉英
Owner SUREXAM BIO TECH
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