Petr A Bai, Anton M Solovjev, Elena A Kubareva, Sergey A Kurzeev, Ivan Yu Sakharov
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引用次数: 0
摘要
开发了一种简单灵敏的测定核酸内切酶（NE）Nt.Bst9I活性的同质-异质和异质检测方法。两个 26 元生物素化 DNA 寡核苷酸的双链体被用作 Nt.Bst9I 的底物。为了提高检测灵敏度，使用了基于链霉亲和素和多过氧化物酶共轭物的化学发光检测系统和增强化学发光反应。所提出的两种检测方法均以微孔板作为固体支持物，从而便于使用 ELISA 设备对 NE 进行自动化分析。通过改变反应介质的酸度、KCl 和 NaCl 的浓度以及温度，找到了有利的条件。尽管所提议的两种检测方法都可用于评估 Nt.Bst9I 的活性，但异质检测方法比同质异构检测方法更灵敏。
Chemiluminescent heterogeneous and homogeneous-heterogeneous assays for determination of nicking endonuclease activity.
Homogeneous-heterogeneous and heterogeneous formats of a simple and sensitive assay for the determination of nicking endonuclease (NE) Nt.Bst9I activity was developed. The duplex of two 26-membered biotinylated DNA oligonucleotides was used as a substrate of Nt.Bst9I. To improve the assay sensitivity the chemiluminescent detection system based on the use of conjugate of streptavidin and polyperoxidase and enhanced chemiluminescence reaction was used. Both proposed assay formats were constructed using microtiter plates as a solid support, allowing for easy automation of NE analysis using ELISA equipment. Varying the acidity, concentration of KCl and NaCl, and temperature of the reaction medium, favorable conditions were found. Although both formats of the proposed assay can be applied to estimate Nt.Bst9I activity, the heterogenous assay was more sensitive than the homogeneous-heterogeneous one.
期刊介绍：
The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field.
The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology.
The journal has been particularly active in:
-Analytical techniques for biological molecules-
Aptamer selection and utilization-
Biosensors-
Chromatography-
Cloning, sequencing and mutagenesis-
Electrochemical methods-
Electrophoresis-
Enzyme characterization methods-
Immunological approaches-
Mass spectrometry of proteins and nucleic acids-
Metabolomics-
Nano level techniques-
Optical spectroscopy in all its forms.
The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.
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