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

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...

AI Technical Summary

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 slightly more complicated than PCR-ASO, and the reagents used are also more expensive

[0004] 2) Allele-specific PCR method: Because PCR requires high complementarity between the 3′ end of the primer and the template, as long as there is one base mismatch in the 3′ end sequence, the amplification efficiency of PCR can be significantly reduced

However, if it is only used for the screening of a few point mutations, its advantages are not obvious

The equipment and reagents used are more expensive

Since this method is based on molecular hybridization of nucleic acids, unknown mutations cannot be detected