Method for detecting DNA intra-molecularly hybridization with known point mutation - polyacrylamide gel electrophoresis

A technology of polyacrylamide gel and DNA molecules, applied in biochemical equipment and methods, analytical materials, measuring devices, etc., can solve the problems of low PCR amplification efficiency, insignificant advantages, and inability to detect unknown mutations

Inactive Publication Date: 2006-01-18
李卫 +1
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Problems solved by technology

However, due to the need to use radioisotopes, there is some damage to the operator's body, and the test results are easily affected by the half-life cycle of radioisotopes
Some scholars have also developed a reverse-phase hybridization method: immobilize specific oligonucleotide probes for various mutations on the same nylon membrane, and then denature the PCR products labeled with biotin or digoxin to be tested. , hybridized with oligonucleotide probes on the nylon membrane, can detect multiple mutations at the same time, but for the detection of a single mutation, the operation of this method is

Method used

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Experimental program
Comparison scheme
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Embodiment approach

[0028] (1) Reagents

[0029] The reagents used, whether imported or domestic, must be of analytical purity.

[0030] (2) Method

[0031] 1) Extract DNA using standard phenol / chloroform method.

[0032] 2) PCR: the β-globin gene was amplified by nested PCR (nested PCR). The amplified fragments and primer sequences are listed in Table 1.

[0033]2.1 Primer design: Primers F, D, C, β54, α8 and α9 are external primers. Fragment FD, fragment Cβ54, and fragment 89 were generated, respectively, and used as templates for internal primer PCR. The other primers are internal primers, and the PCR fragments generated by them are used for IMH-PAGE detection.

[0034] 2.2 Outer primer PCR: the volume of the outer primer PCR mixture is 10 μl. Which contains 50mM KCl, 10mM Tris-HCl (pH8.8 at 25 ℃), 0.08% (volume %) Nonidet P40, 1.5mM MgCl 2 , each dNTP 0.125mM, each outer primer 10pmol, 0.4u Taq DNA polymerase (MBI Fermentas, USA) and 0.2μg genomic DNA. The PCR thermocycling conditions...

Embodiment 1

[0043] The patient, Wei, came to see a doctor because of the birth history of a child with severe β-thalassemia. Take 0.5 ml of venous blood, extract the DNA with the standard phenol / chloroform method, and detect it with the IMH-PAGE method, using aggt as primers t When gagtGTGGACAGATCCCCAAAG was confirmed, the patient carried CDs41-42(-TTCT)β-thalassemia gene point mutation, which was consistent with the detection results by traditional PCR-ASO method.

Embodiment 2

[0045] Patient Li, because of the birth history of a child with severe β-thalassemia, went to the doctor. Take 0.5 ml of venous blood, extract the DNA with the standard phenol / chloroform method, and detect it with the IMH-PAGE method, using tcacct as the primer a At gccccCTGCCGTTACTGCCCTG, the patient was diagnosed as carrying a CD17(A-T)β-thalassemia gene point mutation, which was consistent with the results detected by the traditional PCR-ASO method.

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Abstract

The invention relates to a non-isotope method for testing point mutation, which uses DNA molecule inner hybrid probe and the connected primer. The invention has been completely used to detest known eleven ª‰ thalassemia point mutation and two ª‡ thalassemia point mutation, that is: -28(A-G), -29(A-G), CD17(A-T), CD26(G-A, HbE), CD30(A-G), CDs41/42(-TTCT), CD43(G-T), CDs71/72(+A), CD95(+A), IVS-I-1(G-T)IVS-II-654(C-T), CD125(CTG-CCG, Hb Quong Sze) and terminating codon(TAA-CAA, Hb Constant Spring).

Description

technical field [0001] The invention belongs to the field of non-isotopic methods for detecting gene point mutations in the medical field. Background technique [0002] With the development of medical science, people have already found that many diseases are caused by point mutations in genes; in order to accurately find out the location of point mutations, medical scientists have done a lot of research and achieved remarkable results, such as The detection techniques for known point mutations have been published in the following reports: [0003] 1) Polymerase Chain Reaction-Allele Specific Oligonucleotide Probe Dot Hybridization (Polymerase Chains Reaction-AlleleSpecific Oligonucleotide, PCR-ASO) method: first, the PCR method is used to amplify the target gene fragment, and the amplified product After denaturation, the sample was directly spotted on the nylon membrane, and then mixed with radioactive isotope ( 32 p) The labeled specific oligon...

Claims

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Application Information

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IPC IPC(8): G01N27/447C12Q1/68
Inventor 李卫高枫
Owner 李卫
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