Nucleic acid monitoring methods

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. 

The quantitation of HCV RNA in serum may be important in predicting and monitoring response to antiviral therapy (Davis, 1994). A variety of methods have been used to quantitate HCV RNA, including endpoint dilution RT-PCR, competitive PCR, multicyclic RT-PCR, nucleic acid sequence-based amplification, RT-PCR with a single internal quantitation standard, and bDNA (Chazouilleres et al, 1994 Detmer et al., 1996 Ishiyama et al, 1992 Kaneko et al., 1992 Klevits et al., 1991 Kobayashi etal., 1993 Miskovsky etal., 1996). 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

Nucleic acids can be visualized on the slab gel after separation by soaking in a solution of ethidium bromide, a dye that displays enhanced fluorescence when intercalated between stacked nucleic acid bases. Ethidium bromide may be added directly to the agarose solution before gel formation. This method allows monitoring of nucleic acids during electrophoresis. Irradiation of ethidium bromide-treated gels by UV light results in orange-red bands where nucleic acids are present. 

As an analytical tool, electrophoresis is simple, relatively rapid, and has unparalleled resolving power. It is used chiefly for analysis and purification of very large molecules such as proteins and nucleic acids. Highly sensitive detection methods have been developed to monitor and analyze electrophoretic separations. 

PCR is a very powerful technique, providing a sizable amount of DNA from a trace of a DNA sample. Hence it would be natural to expect that the trace amount of starting DNA can be quantitated sensitively from the amount of the finally-obtained PCR product. However, this type of quantitation based on the end-point detection is not reliable because of the saturation effects of PCR. This problem has been overcome by real-time PCR, which monitors PCR amplification in real time and enables accurate quantitation from the kinetics of the exponential phase. Real-time PCR thereby provides a highly sensitive and specific quantitation method for nucleic acids. 

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