Nicking enzymes assays

Thrombin-binding aptamers have been used to profile thrombin in human blood, to detect thrombin by attachment of the aptamer inside a nanopore, and in an assay that uses DNasel as a nicking enzyme. Assays have been developed to detect thrombin using its aptamers in a variety of methods, including fluorescence, chemiluminescence, ... 

The enzyme without the sigma factor, called core polymerase, retains the capability to synthesize RNA, but it is defective in the ability to bind and initiate transcription at true initiation sites on the DNA. In fact when RNA polymerase was first purified from crude extracts it was missing the a factor. The assay for polymerase involved the use of DNA with single-strand nicks. When a DNA template was used that did not have single-strand nicks, this enzyme was not active. This led to a search for a missing factor. When this factor (cr70) was added back to the purified core enzyme and the uncut DNA template, the enzyme was able to bind... 

TUNEL The TUNEL assay represents a historical low point in attempts to coin acronyms. The Terminal deoxynucleotidyl transferase mediated dC/TP Nick End-Labeling assay labels the ends of DNA with fluorescent UTP. Apoptotic cells often have fragmented DNA these fragments will provide more substrate for the enzyme terminal deoxynucleotidyl transferase. Apoptotic cells can therefore be enumerated by flow cytometry according to their increased intensity in the TUNEL assay. 

TUNEL stands for terminal deoxynucleotidyl transferase x-dUTP nick end labeling. This assay is based on the detection of DNA fragments marked by an enzyme that incorporates modified nucleotides to the 3 -OH ends of the fragments, which can be then specifically detected. The enzyme is a deoxynucleotidyl transferase, which can act in absence of a complementary strand. Among the nucleotides, there is one specifically marked with a fluorochrome, an enzyme, or an antigen. This allows different methods of detection. 

A typical assay, which is commonly adopted by commercial enzyme suppliers, is based on the incorporation of labeled dNTPs by nick translation. The mixture (50 pi) contains 50 mM Tris-Cl (pH 9.0 at 25°C), 50 mM NaCl, 10 mM MgCl2, 0.2 mM each of dATP, dCTP, and dGTP, 50 pM [ H]dTTP, and 12.5 pg activated (DNase I-treated) calf thymus DNA. The mixture is incubated at 74°C. 

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