Method for preparing length relying probe for detecting gene mutation

A length-dependent, long-probe technology, applied in the field of oligonucleotide probe preparation, can solve the problems of cumbersome biological preparation methods, achieve diverse and convenient sources, simple primers, and improve the detection success rate

Inactive Publication Date: 2014-10-15
上海中优医药高科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The object of the present invention is to provide a method for preparing MLPA length-dependent probe pairs with simple operation method, controllable pollution and high preparation efficiency for the above-mentioned MLPA length-dependent probe pair biological preparation method is cumbersome.

Method used

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  • Method for preparing length relying probe for detecting gene mutation
  • Method for preparing length relying probe for detecting gene mutation
  • Method for preparing length relying probe for detecting gene mutation

Examples

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Effect test

Embodiment 1

[0046] Example 1 The preparation and detection of the length-dependent probe pair for detecting the mammalian cardiovascular disease-related gene ACE gene includes the following steps:

[0047] 1. Design two pairs of primers for the ACE gene to be tested. One pair is a universal primer for subsequent PCR amplification detection. They are GSF and GSR respectively. The natural sequence (NS) is irrelevant; the detection primers for the target site or fragment to be detected are TSF and TSR, respectively.

[0048] 2. Prepare the long probe in the length-dependent probe pair:

[0049] 1) Determine a natural sequence (NS) that has nothing to do with the ACE gene to be tested and the universal primer, according to the NS and the sequence of the ACE gene to be tested within the chicken house;

[0050] 2) For long probes, a pair of amplification primers are involved, namely GSR-N' and TSR-N', wherein GSR-N' is composed of the sequence of GSR and the sequence of about 18bp at the 3' en...

Embodiment 2

[0152] Example 2 The preparation and detection of length-dependent probe sets for detection of mammalian cardiovascular disease-related genes ACE, GNB3, and NOS3 genes include the following steps:

[0153] 1. Design a pair of universal primers for the length-dependent probe sets of ACE, GNB3, and NOS3 genes for subsequent PCR amplification detection, which are GSF and GSR respectively. The native sequence (NS) in the probe is irrelevant.

[0154] The concrete nucleotide sequence of above-mentioned primer is:

[0155] GSR: 5'-CACACAGGAAACAGCTATGAC-3' 21bp

[0156] TSR: 5'-ACCTGCTGCCTATACAGTCACTTTTT-3' 25bp

[0157] 2. Design a pair of detection primers for the target site or fragment of the ACE gene to be tested, namely TSF and TSR.

[0158] 3. Design a pair of detection primers for the target site or fragment of the GNB3 gene to be tested, namely TSF1, TSRC1 and TSRT1.

[0159] 4. Design a pair of detection primers for the target site or fragment of the NOS3 gene to be tes...

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Abstract

The invention discloses a method for preparing a length relying probe for detecting gene mutation. The method comprises the following steps: 1) selecting a carrier irrelevant to a target sequence to be detected as a template, and designing an amplimer sequence of a length relying probe pair according to a target nucleotide sequence and an unrelated sequence; 2) manually synthesizing a primer; 3) conducting PCR amplification; 4) purifying and recycling a PCR product; 5) digesting the PCR product by adopting a T7 excision enzyme; 6) recycling ssDNA. The method for preparing the MLPA probe is low in cost, all operations of amplification, enzyme digestion and the like can be finished only through a PCR instrument, dsDNA and nucleotide which are not digested completely are digested and excluded by utilization of the T7 excision enzyme, the probe preparation efficiency is improved, and the hybridization efficiency in the MLPA experiment is improved.

Description

technical field [0001] The invention relates to the preparation of oligonucleotide probes, in particular to the preparation method of length-dependent probes that can be used to detect gene mutations, including single nucleotide polymorphism (SNP), copy number variation (CNV) and chromosome number abnormalities. Background technique [0002] Detecting gene mutations is the main method for diagnosing genetic diseases and an important auxiliary diagnostic method for other metabolic and neurological diseases. Gene mutations include single nucleotide polymorphisms (SNPs), copy number variations (CNVs), gene segment duplications and deletions (Ins / Del), abnormal chromosome number, gene methylation, etc. Among them, single nucleotide polymorphisms (SNPs), copy number variations (CNVs) and chromosome number abnormalities are the main types of gene mutations currently detected. [0003] At present, the technical methods for detecting gene mutations are mainly based on PCR technolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6811C12Q2531/113C12Q2521/319
Inventor 何骏奇毛丹丹王校卞雪莲
Owner 上海中优医药高科技股份有限公司
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