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Method for screening mutation of red transcription factor EKLF gene and application thereof

A gene and detection method technology, which is applied in the field of screening erythroid transcription factor EKLF gene mutation, can solve the problems of cumbersome operation steps, high detection cost, and long time consumption, and achieve the effect of simple operation, high sensitivity, and fast speed

Pending Publication Date: 2021-11-09
GUANGDONG WOMEN & CHILDREN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above problems, the object of the present invention is to provide a detection kit using HRM technology to detect unknown mutations of the EKLF gene, to solve the problem that the current detection reagent materials used in the above-mentioned background technology need to be prepared by many parties, and the experimental operation steps are cumbersome and time-consuming. Longer, easy to pollute, higher detection cost, etc.

Method used

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  • Method for screening mutation of red transcription factor EKLF gene and application thereof
  • Method for screening mutation of red transcription factor EKLF gene and application thereof
  • Method for screening mutation of red transcription factor EKLF gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] According to the EKLF gene sequence in GenBank, the present invention uses Primer Express3.0 software to design 9 pairs of specific primers covering the non-coding region of EKLF gene (promoter region, splicing site, 5'UTR and 3'UTR) And all coding regions (exon 1, exon 2 and exon 3), the shortest amplicon fragment length is 179bp, the longest is 348bp, and its specificity was initially identified by NCBI's BLAST function. The positions of the primers cover each other, avoiding screening "dead spots".

[0043] The 9 pairs of upstream and downstream primers are as follows:

[0044] 1) The primers for the upstream and downstream of the EKLF gene promoter region are: 5'-TACCCAGCACCTGGACCCTC-3', 5'-GAGGCTGTGATAGCCCCTTCG-3';

[0045] 2) The primers for the upstream and downstream of the exon 1 region of the EKLF gene are: 5'-CTAAGGACAGAGAGGAGCCC-3', 5'-CAGCCAGCCCACCTAGAC-3';

[0046] 3) The primers for the upstream and downstream regions of exon 2-1 of the EKLF gene are: 5...

Embodiment 2

[0054] The present invention discloses a reaction system and PCR reaction conditions containing the above-mentioned PCR primer set, wherein the reaction system mainly includes the following components: 9 sets of primer pair mixture (10 μM) described in Example 1, negative quality control substance, 10×Taq buffer (containing 20mM Mg2+), 2.5nM dNTPs, 5U / μl Taq DNA polymerase, 1×LC Green, template DNA, 5M Betain, DMSO and deionized water.

[0055] Wherein the reaction condition is:

[0056] 1) PCR amplification conditions: pre-denaturation at 95°C for 5 minutes; 45 cycles (melting: 95°C for 30 seconds, annealing: 63-68°C for 30 seconds, extension: 72°C for 30 seconds); denaturation: 95°C for 1 minute, Keep warm at 4°C.

[0057] 2) Reaction conditions for HRM analysis with a resolution melting curve analyzer (LightScanner HRⅠ96): the initial temperature "Start Temp" is 55°C, the end temperature "End Temp" is 98°C and the standby temperature "Hold Temp" is 42°C, When the temperat...

Embodiment 3

[0058] Example 3 Taking the mutation screening of EKLF gene promoter region as an example, the HRM detection of EKLF gene mutation was carried out.

[0059] 1) Specifically include the following:

[0060] Step 1, DNA preparation of the sample to be tested: collect the peripheral blood sample of the subject to be tested, and extract the sample DNA by column method;

[0061] Step 2, Aliquot the reaction solution: mix the upstream and downstream primers of the EKLF gene promoter region and the PCR reaction solution, and divide it into a 96-well PCR plate of Bio-Rad, each well includes 1.0μL of 10×Taq buffer (containing 20mM Mg2+) , 2.5nM dNTPs 0.4μL, 5U / μl Taq DNA polymerase 0.2μL, 10μM upstream and downstream primers 0.6μL, 1×LC Green 1μL, template DNA 0.8μL, 5M Betain 2.0μL, DMSO 1.0μL, deionized water 3.0μL;

[0062] Step 3, add the DNA of the sample to be tested: add 1 μL of negative quality control products to the first three wells of the 96-well PCR plate, and add 1 μL of ...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a method for screening mutation of a red transcription factor EKLF gene and application thereof. The method is characterized in that an amplification primer is designed according to an EKLF gene sequence, and screening is conducted on unknown mutation of the EKLF gene in a sample by means of an optimized high-resolution melting curve reaction system. The detection method is easy to operate, high in speed and high in flux, a PCR product does not need to be subjected to aftertreatment, the difference of single basic groups can be detected, closed-tube operation is truly achieved, and the method has wide clinical application prospects.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for screening the mutation of the erythroid transcription factor EKLF gene and an application thereof. Background technique [0002] The erythroid transcription factor EKLF / KLF1 was first discovered by Bieker, a scientist at the Mount Sinai School of Medicine in the United States. It regulates a series of growth and development processes of erythrocytes by specifically recognizing and binding conserved DNA sequences to initiate the transcription of downstream genes. The EKLF gene (NC_000019.10) is located at 19p13.2, with a full length of 2781bp, containing three exons and two introns, and the protein consists of two functional domains: one is located at the N-terminus, which is enriched by proline A transcriptional activation domain; another, located at the C-terminus, is a zinc finger domain that binds conserved DNA sequences. Studies have shown that EKLF gene d...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2527/107C12Q2563/107
Inventor 刘顿余丽华刘风华张曦倩
Owner GUANGDONG WOMEN & CHILDREN HOSPITAL
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