Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof

Abstract

The invention discloses a reverse transcription-polymerase chain reaction (RT-PCR) primer of a human leukemia fusion gene BCR-ABL, which is characterized in that nucleotide sequence of the primer is: BCR_exF: 5'-TTCCGCTGACCATCAATAAG-3'; ABL_exR: 5'-CGCATGAGTTCATAGACCTT-3'; ABL_inF: 5'-CGAGTTGGTTCATCATCATTCA-3'; and ABL_inR: 5'-CCTTCTCTAGCAGCTCATACA-3'. The invention further discloses method for performing RT-PCR by using the human leukemia fusion gene BCR-ABL, a pure single reverse transcription product can be obtained according to the method, a nested polymerase chain reaction is used to achieve qualitative detection of existence of the BCR-ABL and amplify segments used for detecting sequence and containing fourteen common ABL kinase point mutational sites, and the method can remarkably improve amplification success rate of the human leukemia fusion gene BCR-ABL.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a methodological improvement of clinical testing technology, which uses specific reverse transcription (RT) primers to reverse transcribe the p210 protein encoded by the human leukemia fusion gene BCR-ABL mRNA, a 1.5kb fragment including the sequence of the ABL kinase domain. The amplification success rate of RT-PCR can be significantly improved. Background technique [0002] Chronic myeloid leukemia (CML) is a malignant clonal proliferative disease of the blood system that occurs in hematopoietic stem cells. 95% of the abnormal white blood cells of CML patients contain an abnormal chromosome named "Philadelphia" formed by chromosomal translocation. Its formation mechanism is that a section of chromosome 9 is spliced ​​to chromosome 22, and this splicing forms a new abnormal gene BCR-ABL with carcinogenic effect. The protein encoded by the BCR-ABL gene has abnorma...

AI Technical Summary

Problems solved by technology

The RT-PCR part of the project has the following two difficulties in the technical link: 1. The project needs to extract the mRNA in the blood leukocytes. Due to the many uncontrollable factors in the logistics and transportation of clinical specimens, the quality of the clinical test is often not obtained. Excellent whole blood samples, so there will be large sample-to-sample variability in the quality of mRNA obtained

2. Since the splicing point of the fusion gene is far away from the ABL kinase region to be detected, the fragment that needs to be amplified by RT-PCR is relatively large, about 1.5kb, so the reverse transcription process requires high requirements

Although the cDNA products obtained by random primers contain a large amount of fragment information and can be used for cloning various genome sequences, the specificity of random primers is not good, and for some genes with low mRNA copy number, the effect of reverse transcription is not ideal, which may lead to Subsequent PCR amplification fails, or produces nonspecific products

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