Assay of nucleic acids

Electrochemical assays of nucleic acids based on DNA hybridisation have received considerable attention [34,57-60]. DNA hybridisation biosensors are a very attractive topic in the clinical diagnostics of inherited diseases and the rapid detection of infectious microorganisms. 

The sensitivity of the detection is usually improved by the silver enhancement method. A better detection limit was reported when a silver enhancement method was employed, based on the precipitation of silver on AuNPs tags and its dissolution (in HNO3) and subsequent electrochemical potentiometric stripping detection [43]. The new silver-enhanced colloidal gold stripping detection strategy represented an attractive alternative to indirect optical affinity assays of nucleic acids and other biomolecules. 

Miller, Herbert K., Microbiological Assay of Nucleic Acids and Their... 

Figure 2-17. Reactions involved in the assay of nucleic acids by the orcinol procedure.

Vol. XIIB [174]. Preparation and Assay of Nucleic Acids as Antigens. O. J. Plescia. 

The interactions of nucleic acid materials with Ln have been largely studied through their luminescent properties. The known affinity of Ln " cations for the phosphate moiety has led to many investigations of the Ln interactions with nucleotides, covered in some detail by Evans [1]. The interaction of Ln with nucleic acids has been used to probe perturbations in the integrity of nucleic acid strands and as an assay of nucleic acid hybridization. Balcarova and Brabec [45] showed that the interaction of Tb with double-stranded DNA can be used to monitor guanine bases present in distorted double-stranded regions of DNA. The enhancement of distortions in double-stranded DNA appears to be restricted to certain modifiers of nucleic acid as there is no... 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

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. 

Current analytical methods have difficulty detecting picogram levels of nucleic acids, particularly when high levels of other biopolymers (e.g., proteins) are present. The most widely used assay method employed by the pharmaceutical industry involves a nick translation DNA hybridization method (1). This assay offers high sensitivity and selectivity but has a number of drawbacks. 

The reference standards are used to quantitate the standards that are employed in the kits to generate the standard curves. The kit standards are recombinant single-stranded DNA molecules that are added to either negative serum or plasma at known concentrations. Because the standard curve is not constructed with reference standards, Chiron initially chose to use the term equivalent to describe the units of nucleic acid quantitation in clinical samples. An equivalent was defined as the amount of nucleic acid in a clinical sample that gave a signal equal to one molecule of the reference standard nucleic acid. The term copy rather than equivalent is used to describe the units of nucleic acid quantitation in the HIV-1 bDNA assay. The terms are now used interchangeably. 

Collins, M. L et al. (1997). A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res. 25,2979-2984. 

Overall, the significant Stokes shift of 100 nm and the good quantum yields make the coumarin dye a powerful fluorescent probe for nucleic acids assays or cell biology. The postsynthetic click chemistry makes this fluorophore readily accessible for fluorescent labeling of nucleic acids. 

Similar techniques can be used to devise (strept)avidin-biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind... 

The chemical modification of nucleic acids at specific sites within individual nucleotides or within oligonucleotides allows various labels to be incorporated into DNA or RNA probes. This labeling process can produce conjugates having sensitive detection properties for the localization or quantification of oligo binding to a complementary strand using hybridization assays. 

The chemistry, metabolism, and clinical importance of folic acid have been the subject of many excellent reviews (A7, Gil, H14, H20, Rl). Folic acid deficiency leads to a macrocytic anemia and leucopenia. These symptoms are due to inadequate synthesis of nucleic acid. The synthesis of purine bases and of thymine, required for nucleic acid synthesis, is impaired in folic acid deficiency. Detection of folic acid activity in biologic fluids and tissues is of the utmost importance it distinguishes between the various anemias, e.g., those due to vitamin Bi2 or folic acid deficiency. Because morphology of the abnormal red cell does not help in diagnosing vitamin deficiency, one must rely on assay methods for differential diagnosis. Treatment of pernicious anemia with folic acid has led to subacute combined degeneration of the spinal cord despite... 

Huhtinen P, Vaamo J, Soukka T et al (2004) Europium(III) nanoparticle-label-based assay for the detection of nucleic acids. Nanotechnology 15 1708-1715... 

The Polymerase Chain Reaction. In the past, a major drawback of hybridization assays was their need for relatively large amounts of sample DNA to compensate for their low sensitivity. This problem has been surmounted in recent years by the development of powerful enzymatic techniques that can exponentially replicate specific DNA sequences in the test tube. With these techniques it is now possible to analyze vanishingly small samples that initially contain fewer than 10 copies of the sequence of interest. The new methods take advantage of the chemical properties of nucleic acids and of highly specialized enzymes that can repair and replicate DNA in vitro. 

The reader is referred to other reviews for detailed discussions of the electronic states and luminescence of nucleic acids and their constituents/0 fluorescence correlation spectroscopy/2) spectroscopy of dye/DNA complexes/0 and ethidium fluorescence assays/4,0 A brief review of early work on DNA dynamics as well as a review of tRNA kinetics and dynamics have also appeared. The diverse and voluminous literature on the use of fluorescence techniques to assay the binding of proteins and antitumor drugs to nucleic acids and on the use of fluorescent DNA/dye complexes in cytometry and cytochemistry lies entirely outside the scope of this chapter. 

In this chapter, we will survey the kinds of solid supports (substrates) and surface chemistries currently used in the creation of nucleic acid and protein microarrays. Which are the best supports and methods of attachment for nucleic acids or proteins Does it make sense to use the same attachment chemistry or substrate format for these biomolecules In order to begin to understand these kinds of questions, it is important to briefly review how such biomolecules were attached in the past to other solid supports such as affinity chromatography media, membranes, and enzyme-linked immxm-osorbent assay (ELISA) microtiter plates. However, the microarray substrate does not share certain unique properties and metrics with its predecessors. Principal among these are printing, spot morphology, and image analysis they are the subjects of subsequent chapters. 

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

Haworth R, Pilling AM. The PCR assay in the preclinical safety evaluation of nucleic acid medicines. Human Experi Toxicol 19.5 (200) 267-276. 

To understand how these modem methods work, it is necessary first to review some general laboratory techniques ubiquitous in genetic engineering. Among the most important are gel electrophoresis of nucleic acids, nucleic acid hybridization assays, and the polymerase chain reaction. 

Currently, there is a need for high-throughput determination of nucleic acid sequences. At present, detection systems most commonly employ fluorescence-based methods. However, wide spread applications of such methods are limited by low speed, high cost, size, and number of incubations steps, among other factors. Application of electrochemical methods in affinity DNA sensors presents likely a promising alternative, allowing miniaturization and cost reduction, and potentially allowing application in point-of-care assays. 

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