Specific primer and liquid phase chip for BRAF genetic mutation detection

A technology of specificity and detection solution, applied in the field of molecular biology, can solve the problems of false positive rate, poor timeliness, low sensitivity, etc., and achieve the effect of high detection specificity and accuracy, stable and reliable effect

Active Publication Date: 2011-11-09
SUREXAM BIO TECH
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AI Technical Summary

Problems solved by technology

The PCR-sequencing method has the advantage of being able to determine the range and type of mutations, and is currently a widely used detection method, but the sensitivity of the sequencing method is only 20%-25%, which is far from meeting the needs of practical applications, especially for heterogeneity of tumor somatic mutations, low sensitivity will lead to a large number of missed detections
At the same time, the detection operation of the sequencing method is complicated and the timeliness is poor. For the detection of practical applications that require high timeliness and high sensitivity, the limitations of the sequencing method have long been highlighted
Real-time fluorescence quantitative PCR detection technology has high detection efficiency and strong timeliness, but

Method used

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  • Specific primer and liquid phase chip for BRAF genetic mutation detection
  • Specific primer and liquid phase chip for BRAF genetic mutation detection
  • Specific primer and liquid phase chip for BRAF genetic mutation detection

Examples

Experimental program
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Effect test

Embodiment 1B

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

[0021] 1. ASPE Primers

[0022] Aiming at the V600E mutation site of BRAF, the wild-type and mutant ASPE primer pairs were designed respectively.

[0023] The design points of ASPE primers for BRAF gene mutation detection are as follows:

[0024] ASPE primers consist of "Tag + specific primer sequence". Among them, the 5' end is the Tag sequence designed according to the BRAF gene mutation detection. The designed Tag sequence can avoid the secondary structure that may be formed by the ASPE primer in the reaction system to the greatest extent, and the Tag sequence and the Tag sequence, the Tag sequence and the specific There is no cross-reaction between the primer sequences. The Tag sequence and the specific primer sequence form complete ASPE primers, and enable all ASPE primers to react synchronously in a uniform reaction system (that is, the buffer environment of the same reaction, the same rea...

Embodiment 2

[0053] Example 2 Detection of samples using BRAF gene mutation detection liquid chip

[0054] The formula of described various solutions is as follows:

[0055] 50mM MES buffer (pH5.0) formula (250ml):

[0056] Reagent

[0057] 2×Tm hybridization buffer

[0058] Reagent

[0059] Store at 4°C after filtration. ExoSAP-IT kit was purchased from US USB Company.

[0060] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0061] 1. Sample DNA extraction:

[0062] Refer to the DNA extraction method in "Molecular Cloning" to obtain the DNA to be detected.

[0063] 2. PCR amplification of samples to be tested

[0064] Primers were designed using Primer5.0, and the target sequence containing the detection site was amplified by PCR, and the product size was 190bp. The primer sequences (SEQ NO.37-38) are shown in Table 7 above.

[0065] First prepare the PCR primer working solution: take 100ul of the stock solut...

Embodiment 3

[0118] The selection of embodiment 3 wild-type and mutant-type specific primer sequences

[0119] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0120] Taking the detection liquid chip of BRAF gene V600E site mutation as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the V600E wild type, and the specific primer sequence was derived from SEQ ID NO.1 and SEQ ID NO.13, respectively. The specific primer sequences at the 3' ends of the mutant ASPE primers were respectively derived from SEQ ID NO.2 and SEQ ID NO.14, and the Tag sequences at the 5' ends of the wild-type and mutant ASPE primers were fixed as SEQ ID NO.25 and SEQ ID NO.25 respectively. SEQ ID NO.26, correspondingly, the anti-tag sequences coated on the microspheres and complementary to the corresponding tag sequences are SEQ ID NO.31 and SEQ ID NO.32, respectively. The specific design is shown in the following table...

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Abstract

The invention discloses a liquid phase chip for BRAF genetic mutation detection. The liquid phase chip mainly comprises (A) respectively designed ASPE primer pairs in the wild type and in the mutant type directed to a V600E mutation site of the BRAF: each ASPE primer is composed of a tag sequence at a 5'terminus and a specific primer which is at a 3'terminus and directed to a target gene mutation site, one base of last three bases at the 3'terminus of the specific primer is the mutation site, and the Tm value of the specific primer is from 52 to 58 DEG C; (B) two types of microspheres which have color-different codes and are coated with specific anti-tag sequences which can correspondingly have complementary pairing with the selected tag sequence in the (A); and an amplimer for amplifying the target sequence of the BRAF gene having the V600E mutation site. The liquid phase chip for BRAF genetic mutation detection prepared in the invention has an excellent signal to noise ratio, and a cross reaction does not substantially exist between a designed probe and the anti-tag sequence.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to specific primers for BRAF gene mutation detection and a liquid phase chip. technical background [0002] Murine sarcoma viral oncogene homolog B1 (v-taf moourine sarcoma viral oncogene homolog B1, BRAF) gene is located on human chromosome 7q34, and its functional coding region consists of 2510 base pairs, encoding the MAPK pathway The serine-threonine-protein kinase in the cell transduces signals from Ras to MEK1 / 2, thereby participating in the regulation of various biological events in cells. Studies have shown that BRAF mainly has two types of mutations: ① 11% of the mutations are located in the glycine loop on exon 11, such as G463, G465, G468 and other point mutations; ② 89% of the mutations occur in the activation region of exon 15, About 92% of them are located at the 1799th nucleotide (T is mutated to A), resulting in the substitutio...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 许嘉森李国强余刚秦会娟
Owner SUREXAM BIO TECH
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