Methods of DNA Detection

Methods of DNA manipulation now make it possible to insert DNA into prokaryotic, eukaryotic, or viral hosts, creating versatile marker systems that allow as- sessment of the survival and spread of strains, studies on gene transfer, and determinations of cell activity. A potential marker gene must be absent from the strain used in the study, and either absent or in sufficiently low abundance in the microbial population under study to allow detection of marked cells at an appropriate level. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Current methods for DNA detection usually require enzymatic amplification of the target DNA sequence prior to analysis. For example, the PCR technique selectively increases the concentration of the target sequence relative to unrelated sequences. PCR methods, however, introduce ambiguities resulting from contamination by different DNA sequence. Therefore, a definitive method is required for the analysis of a single, original DNA sequence. To achieve this objective, the sensitivity and speed of the chemiluminescent enhancement techniques described in this chapter must be improved. 

Unfortunately, direct electrochemical detection of DNA damage in films suffered from poor signal to noise ratios and data analysis that required derivative or other background corrections. Thus we explored catalytic methods of DNA oxidation (cf. Eqs. 3 and 4) to improve signal to noise in SWV detection.[45] At the same time, we began to realize that layer-by-layer growth of films had... 

The more sensitive and convenient method of PolyP detection in situ is fluorescence microscopy using fluorochromes of the type 4/,6/-diamino-2-phenylindole.2HCl (DAPI), which is commonly used for DNA detection. At a high concentration (50 mg ml 1), it also stains PolyP granules and lipid inclusions (Allan and Miller, 1980 Tijssen et al, 1982 Streichan et al, 1990). DAPI-DNA fluorescence is blue-white, while DAPI-PolyP and DAPI-lipid fluorescence is yellow. The lipid fluorescence is weak and fades in a few seconds, while the PolyP granules appear bright yellow, thus allowing discrimination of the above types of cell inclusions (Streichan et al, 1990). 

An efficient method for DNA detection without a fluorescent dye on the nanoscale can be realized through the use of a single light-emitting polymer NW with lightly doped states, as reported by Park and coworkers [139]. Biological materials can be... 

There are two basic strategies by which this might be accomplished. The first is to develop the chemistry for using fluorescence detection in conjunction with the chemical method of DNA sequencing. In contrast to the enz3nnatlc method, the chemical... 

Physical methods of DSB detection are based on changes in the size-sensitive properties of the DNA molecule. Although these methods allow very straightforward and precise DNA size determinations for relatively small molecules (up to 100 kbp), they frequently fail when applied to DNA molecules the size of human chromosomes (average of 150 Mbp). 

Other applications of nanoparticles for SPRi enhancement are the original method for DNA detection by He et al. (22), and the detection of microRNAs (miRNAs) down to a concentration of 10 fM using a surface poly(A) polymerase detection and nanoparticle amplification (24). A recent application is the detection of polymerase products in the attomolar range by template-directed polymerase extension of a surface array element with nanoparticle-enhanced detection of the reaction product (25). 

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