Measurement of nucleic acids

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

Mimro, H.N. and Fleck, A. (1966). Recent developments in the measurements of nucleic acids in biological materials. A supplementary review. The Analyst, 91 77-88. 

B 10. What biomolecules would interfere with the measurement of nucleic acids using the spectrophotometric (Am]) assay ... 

This method provided a simple and fast procedure to identify the presence of a nucleic acid sequence with good sensitivity. On average, the liposomes entrapped several thousand dye molecules, which provided intrinsic amplification of individual binding events. A qualitative measure of nucleic acid hybridization by visualization of the colour on the dip stick was intrinsically provided and quantitative measurements were also possible by use of a reflectometer. This approach resulted in a limit of detection of 1 fmol and a dynamic range of over two orders of magnitude. Maximum signal-to-noise was obtained with 0.2 mol% of probe sequence immobilized on the liposomes. 

However, flow injection direct amperometric detection at carbon paste electrodes [42] was used for measurements of nucleic acids down to the 0.5 ng level without time-consuming derivatization or hydrolysis steps. 

Wang, J., Bollo, S., Paz, J. L. L., Sahlin, E., Mukherjee, B. (1999). Ultratrace measurements of nucleic acids by baseline-corrected adsorptive stripping square-wave voltammetry. AwaZ C/iem 71, 1910-1913. 

Since the instrument is measuring transmitted light, it is important that the total absorbance of the cell, sample, and solvent be kept below 1.0. Indeed, an absorbance of 0.87 gives the optimal signal-to-noise ratio. Transparent solvents and short pathlengths are the key to extending CD measurements of nucleic acids to wavelengths shorter than 220 nm. One hundred il of sample at a concentration of 1 mg ml" in 10 mM phosphate buffer will reach an absorbance of 1.0 at 178 nm in commercially available 50 pm cells. 

Subsequently, Schneider (94) suggested the addition of the hot trichloroacetic acid extraction step to the acid-soluble fraction remaining at the end of the ST procedure, thus permitting more direct isolation of the DNA fraction. This procedure (the Schmidt-Thannhauser-Schneider, or STS, procedure), which permits the use of either phosphorus or sugar assays as the measure of nucleic acid, has become a standard rapid tissue assay. Certain refinements necessary for conversion of a tissue assay method to an isotope assay method, involving more precise segregation of cellular constituents, can be regarded as- extensions or adaptations of the basic procedure. For these reasons, the procedure is described in detail. 

We now describe a new approach to measurement of nucleic acid hybridization which does not depend on a spectral change in the fluorophores. The intensity change is due to localization near a thin metal surface by the binding reaction. We examined SPCE for fluorophore-labeled DNA oligomers, which were complementary (ssDNA-Cy3, Figure 24) or not complementary (ssDNA-Cy5) to a surface-bound capture oligomer [43]. 

Perez, J.E. and P.J. Reeds, 1998. A new stable isotope method enables the simultaneous measurement of nucleic acid and protein synthesis in vivo in mice. J. Nutr. 128, 1562-1569. 

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