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PCR (Polymerase Chain Reaction) primer, kit and liquid phase chip for detecting ALK (Anaplastic Lymphoma Kinase) fusion gene

A technology of fusion gene and liquid phase chip, applied in the field of molecular biology, can solve the problems of inability to accurately obtain the fusion type, time-consuming and laborious, and low sensitivity, so as to improve the detection accuracy, overcome low sensitivity and preciseness Analyzing the Effects of Features

Active Publication Date: 2014-05-21
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is relatively intuitive, the test process is too cumbersome, requiring a wide variety of reagents, time-consuming and labor-intensive, and the test results need to be interpreted by experienced experts. The interpretation of the results is relatively subjective, and it can only detect whether the fusion gene is Existence, the specific fusion type cannot be accurately obtained, and the sensitivity is low, which limits the application of this method to a certain extent

Method used

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  • PCR (Polymerase Chain Reaction) primer, kit and liquid phase chip for detecting ALK (Anaplastic Lymphoma Kinase) fusion gene
  • PCR (Polymerase Chain Reaction) primer, kit and liquid phase chip for detecting ALK (Anaplastic Lymphoma Kinase) fusion gene
  • PCR (Polymerase Chain Reaction) primer, kit and liquid phase chip for detecting ALK (Anaplastic Lymphoma Kinase) fusion gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 A kind of PCR primer and positive control substance detected by the fusion gene detection of ALK fusion gene

[0035] 1. RT-PCR amplification

[0036] 1. Reverse transcription

[0037] For the KIF5B-ALK, KLC1-ALK, and TFG-ALK fusion gene subtypes detected by various targets, the present invention first uses random primers to reverse-transcribe the target detection samples, and reverse-transcribes the mRNA in the samples into cDNA. For specific experimental steps, refer to "Molecular Cloning Experiment Guide", the use of random primers for reverse transcription of mRNA is a routine procedure.

[0038] 2. PCR amplification

[0039] The fusion types in Table 1 are the five ALK fusion gene subtypes detected by the target of the present invention. According to the nucleic acid composition characteristics of the fusion gene sequence, the forward primer and reverse primer were designed using Primer5.0. Using the cDNA obtained in step 1 as a template, the 5 targ...

Embodiment 2

[0050] Example 2 uses PCR primers to detect ALK fusion gene in Example 1

[0051] 1. PCR amplification

[0052] Use the PCR amplification primers described in Table 1 to perform PCR amplification on the mRNA reverse transcription product of the target detection sample and the positive control substance to realize the parallel amplification of the target sequences of the five fusion gene subtypes, and to amplify the five fusion gene subtypes in one step. The strip contains the target sequence of the fusion gene subtype, the product size is shown in Table 1, and the amplification primer sequences (SEQ ID NO.1~SEQ ID NO.6) are shown in the above Table 1.

[0053] First, prepare the PCR primer working solution: take 100ul of the primer stock solutions of SEQ ID NO.1~SEQ ID NO.6 respectively in 1.5ml microcentrifuge tubes, and mix well to obtain the multiplex PCR primer working solution. The multiplex PCR reaction system is as follows:

[0054]

[0055] The PCR amplification p...

Embodiment 3

[0059] Example 3 A liquid chip for detecting ALK fusion gene

[0060] 1. ASPE Primers

[0061] Specific primer sequences designed for target detection KIF5B-ALK K15; DEL 15A20, K17; A20; KLC1-ALK K9; A20; TFG-ALKT6; A20, T3; A20 total 5 fusion subtypes. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0062] Table 3 ASPE Primer Sequence (Specific Primer Sequence)

[0063]

[0064] Table 4 ASPE primer sequence (tag sequence)

[0065] serial number

[0066] 9

[0067] 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 sequence (as shown in Table 3 above). All ASPE primers were synthesized by Invitrogen. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0068] 2. Microspheres coated with anti-tag...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) primer, a kit and a liquid phase chip for detecting an ALK (Anaplastic Lymphoma Kinase) fusion gene. The liquid phase chip comprises a PCR amplification primer, ASPE (Allele Specific Primer Extension) primers and microspheres, wherein the ASPE primers consist of tag sequences and specific primers, wherein the sequences of the specific primers are SEQ ID NO.12 for K15; DEL15A20, SEQ ID NO.13 for K17; A20, SEQ IDNO.14 for K9; A20, SEQ ID NO.15 for T6; A20 and / or SEQ ID NO.16 for T3; A20. The liquid phase chip disclosed by the invention has a very good signal noise ratio and can amplify five fusion subtypes by a single step, and the specific primers have very good specificity.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a PCR primer, a kit and a liquid phase chip for detecting ALK fusion gene. technical background [0002] The ALK gene is located at the chromosome 2p23 site. Under normal circumstances, human-derived alk can be transcribed to produce a 6222bp mRNA, which consists of 29 exons and encodes a 1620 amino acid sequence of 200KDa type I transmembrane protein ALK, which is a Receptor tyrosine kinase (receptortyrosinekinase, RTK), is a member of the RTK insulin superfamily. The complete ALK has a typical RTK three-part structure, namely extracellular domain, lipophilic transmembrane domain and intracytoplasmic tyrosine kinase. According to reports in the literature, ALK protein can be expressed in 60% to 85% of primary systemic ALCL, except in a very small number of diffuse large B-cell lymphomas. It is a relatively specific immune system for primar...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6827C12Q1/686C12Q2563/149C12Q2531/113
Inventor 陈昌华陈菲许昌有
Owner SUREXAM BIO TECH
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