分子生物学
清脆的
生物
反式激活crRNA
基因
劈理(地质)
蛋白酶
慢性淋巴细胞白血病
化学
计算生物学
白血病
遗传学
生物化学
酶
Cas9
古生物学
断裂(地质)
作者
Hongbo Li,Weihua Zhao,Jiamei Pu,Shengliang Zhong,Suqin Wang,Ru‐Qin Yu
标识
DOI:10.1016/j.bios.2021.113941
摘要
One of the causes of B lymphocytic leukemia is abnormal expression of the Pax-5a gene. Detection of the Pax-5a gene can provide effective technical means for early screening of B lymphocytic leukemia. In this work, we designed a sensing scheme to detect the Pax-5a gene based on the signal amplification system, which is based on dual-enzyme assisted target gene circulation, and the disordered cleavage of CRISPR/Cas12a protease. The hairpin probe (HP) in this scheme not only contains the binding sites of the target gene and the primer, but also cleverly contains half of the Nt.BbvCI splicing sites. When the target gene is present, through the synergistic effect of KF and Nt.BbvCI, a large number of single strands of a specific sequence can be produced. At the same time, the target gene falls off from the first hairpin and opens the other hairpin to realize the cycle of the target gene. The resulting single-strand can bind to the Cas12a/crRNA binary complex and unlock the anti-cleavage activity of the CRISPR/Cas12a protease. The single strands labeled with the fluorescent group (FAM) and the quenching group (BHQ) around the solution are cleaved, the fluorescence signal of FAM is restored, and a detectable fluorescence signal is generated. The detection limit is as low as 6.77 fM, and the target gene and the mismatch sequence can also be distinguished well. Therefore, the sensing scheme provides a new detection direction for the early diagnosis and screening of B lymphocytic leukemia.
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