Runx1 gene fragmentation and copy number increase detection kit and preparation method thereof

Abstract

The invention belongs to the technical field of RUNX1 gene splitting and copy number increase detection and provides a RUNX1 gene splitting and copy number increase detection kit and a preparation method thereof. A probe for detecting gene splitting is designed according to the characteristic of gene splitting. The preparation method is characterized by selecting RP11-177L11BAC and RP11-77I17BAC clones and respectively marking RP11-177L11BAC and RP11-77I17BAC with green and red to serve as probe sequences, carrying out FISH (fluorescence in situ hybridization) on the sequences and a cell to be detected after purifying, precipitating and dissolving the sequences, and carrying out fluorescent microscopic observation on cell signals, wherein normal somatic cells have two yellow signals; once genes are split, one red signal, one green signal and one yellow signal appear, and the corresponding quantity of yellow signals appear when the copy number increases. By adopting the kit and the preparation method, the method for accurately detecting gene splitting is created, the defects of the existing gene splitting detection means are overcome, the RUNX1 gene splitting and copy number increase incidents are rapidly and accurately detected and identified, and partner genes fused with an RUNX1 gene are determined in combination with subsequent RACE (rapid amplification of cDNA ends) detection.

Description

technical field [0001] The invention belongs to the technical field of detection methods for RUNX1 gene breakage and copy number increase, and specifically relates to a RUNX1 gene breakage and copy number increase detection kit and a preparation method thereof. Background technique [0002] Breakage or copy number increase of the RUNX1 gene can be found in many diseases, and the RUNX1 gene is also called the AML1 gene. Studies have found that 15%-40% of partially differentiated acute myeloid leukemia (acutemyeloidleukemia, AML) (AML-M2) have RUNX1 gene breaks, and fusion with different partner genes to form new fusion genes. The formation of fusion gene can make the location of oncogene move and be activated, which is one of the reasons for tumorigenesis. The current methods for detecting gene breaks mainly include G / R banding karyotype analysis and RT-PCR detection technology based on mRNA. G / R banding karyotype analysis technology cannot detect small chromosome variation...

AI Technical Summary

Problems solved by technology

G / R banding karyotype analysis technology cannot detect small chromosome variation and gene duplication; RT-PCR technology has good sensitivity and is currently the main method for fusion gene detection, but due to the diversity of fusion gene break sites and the "partner" "The complexity of the gene makes it impossible for the conventional RT-PCR method to detect the break fusion of the new unknown gene

FISH technology is also a common method for detecting gene break fusion, but the probes used in current FISH depend on the known situation of the fusion gene. For example, the commercial probe currently used to detect RUNX1 gene break is AML1 / ETO double fusion probe, only Suitable for the detection of RUNX1 and ETO gene break fusion, not suitable for the screening of RUNX1 gene breaks

[0003] In view of the diversity and complexity of fusion genes and the insufficiency of existing detection methods, after analysis and comparison of a large number of reported fusion gene breakage sites, it was found that: although the RUNX1 gene can be combined with different "partner" genes and the same "partner" gene Different breakpoints of different fusion genes form many different fusion genes, but their own breakpoints are relatively constant

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