PML/RARA (promyelocytic leukemia/retinoic acid receptor alpha) fusion gene quick detection probe with low cost and preparation method and application thereof

Abstract

The invention belongs to the field of molecule biology, and particularly relates to a PML / RARA (promyelocytic leukemia / retinoic acid receptor alpha) fusion gene quick detection probe with low cost and a preparation method thereof. The preparation method comprises the following steps of downloading non-repeated sequences of g genomeene groups in the area covered by PML and RARA gene probes from USCS Genome Browser, and importing the segmented sequences into OligoArray software by batches to design the probes; adding a tag sequence onto the designed probe, and directly synthesizing; using a tag primer with a fluorophorefluorescent gene to amplify and mark the probe, so as to obtain the PML / RARA fusion gene quick detection probe. The prepared PML / RARA fusion gene quick detection probe has the advantages that the hybridizing time is short, the hybridizing experiment can be completed within 15 to 30min, the hybridizing signal is bright, the signal to noise ratio is high, the preparation cost is low, and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a low-cost rapid detection probe of PML / RARA fusion gene and its preparation method and application. Background technique [0002] Detection of PML / RARA fusion gene is one of the most specific and sensitive methods for diagnosing acute promyelocytic leukemia (APL), and it is also the most reliable indicator for APL treatment options, curative effect analysis and prognosis analysis. At present, karyotype analysis, PCR method and FISH method are several common detection methods. FISH has the advantages of safety, rapidity, high sensitivity and simultaneous display of multiple colors. [0003] Fluorescence in situ hybridization is a non-radioactive molecular genetic technique developed on the basis of radioactive in situ hybridization in the 1980s. It is a new in situ hybridization method formed by replacing radioactive isotope labels with fluorescein labels. At presen...

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Problems solved by technology

At present, the commonly used detection probes for FISH are mainly marked by the gap translation method or random primer method on specific human genome BAC clones, and then used for in situ hybridization experiments after being blocked by COT-1 DNA, but this method contains a large number of repetitive sequences. COT-1DNA is blocked but the hybridization result still has a high background, and the probes labeled by this method usually need 12-16h (overnight) to complete the hybridization experiment, which is time-consuming and laborious, and this method requires a large amount (usually 50% of the probe concentration) ~100 times) expensive COT-1 DNA for repeat blocking, which greatly increases the cost of probe preparation

In addition, in recent years, a rapid fluorescent in situ hybridization probe preparation method without repetitive sequences has also been established (1h to complete the hybridization experiment), but this method requires the design of a large number of primers or the construction of a large number of vectors to realize the preparation of the probe. Moreover, traditional methods such as gap translation or random primers are still used in the preparation process of probes, resulting in instability between batches; in addition, this preparation method still needs to rely on human genome sequence or BAC clone sequence, and the source of raw materials has certain limitations, and The whole preparation process is cumbersome, and the preparation cost and uncontrollable factors are greatly increased

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