Denaturated single-stranded DNA

Denatured single-stranded DNA can be renatured (annealed) if the denatuiit condition is slowly removed. For example, if a solution containing heat-denatured DNA is slowly cooled, the two complementary strands can become base-paired again (Figure I-l-l 1). 

Denatured single-stranded DNAs from two species can form a hybrid duplex, the degree of hybridization depending on the extent of sequence similarity. Hybridization is the basis for important techniques used to study and isolate specific genes and RNAs. 

In this experiment, we follow the change in the viscosity of a DNA solution when we change the pH of the solution from the very acidic (pH 2.0) to very basic (pH 12.0). At extreme pH values, we expect that the hydrogen bonds will break and the double helix will become single-stranded random coils. A change in the viscosity will tell at what pH this happens. We shall also determine whether two acid-denatured single-stranded DNA molecules can refold themselves into a double helix when we neutralize the denaturing acid. 

Tensammetry has been widely used for studying the behavior of polynucleotides at the DME [88]. The double-helical DNA (native) is adsorbed at the DME in the region —0.2 and —0.9 V vs. SCE. A pronounced desorption peak appears at —1.1 V (Fig. 38 curve 2). The denatured single-stranded DNA (curve 3) is more strongly adsorbed due to higher adsorbability of DNA bases. Two peaks are observed, the peak at —1.1 V is believed to correspond to an interfacial reorientation of adsorbed DNA segments, whereas the more negative peak (at... 

Electrochemical oxidation of thermally denatured single-stranded DNA (ssDNA) was studied on a room temperature ionic liquid N-butylpyridinium hexafluorophosphate (BPPFg) modified carbon paste electrode (IL-CPE) by Sun et al. [27]. The presence of IL layer on the surface of CPE showed the ability of ionic conductivity and cation exchange extraction. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

If DNA from two different species are mixed, denatured, and allowed to cool slowly so that reannealing can occur, artificial hybrid duplexes may form, provided the DNA from one species is similar in nucleotide sequence to the DNA of the other. The degree of hybridization is a measure of the sequence similarity or relatedness between the two species. Depending on the conditions of the experiment, about 25% of the DNA from a human forms hybrids with mouse DNA, implying that some of the nucleotide sequences (genes) in humans are very similar to those in mice. Mixed RNA DNA hybrids can be created in vitro if single-stranded DNA is allowed to anneal with RNA copies of itself, such as those formed when genes are transcribed into mRNA molecules. 

Double-stranded DNA (calf thymus) Single-stranded DNA Heat-denatured DNA Closed circular DNA Ribosomal RNA (rat liver)... 

A protein induced after coliphage N4 infection has been studied. Although it has one or two tryptophans, its intrinsic fluorescence is dominated by the ten tyrosines/1111 Tryptophan fluorescence is seen after denaturing the protein. Upon binding to single-stranded DNA, the tyrosine fluorescence is quenched. This signal has been used to demonstrate that the binding affinity is very dependent on salt concentration and is also very sensitive to the nucleotide sequence. 

DNA probes are radioactively labeled single-stranded DNA moleoiles that are able to specifically hybridize (anneal) to particular denatured DNA sequences. Different kinds of probes have been developed for the recognition of particular genes (Table 1-7-2 and Figure 1-7-8). 

There are cnrrently two sources of monoclonal antibodies to bromodeoxynridine Becton Dickinson Immunocytochemistry Systems and Boehringer Mannheim Biochemicals. Both antibodies have worked eqnally well for the anthor, and both are directed against single-stranded DNA, therefore necessitating the removal of DNA-associated histones and denaturation of the donble-stranded DNA. 

The detection of HIV-related proteins is one of the most challenging tasks. This is especially true because AIDS should be diagnosed as early as possible to enable an early and effective therapy of this infection. Pavski and Le (57) used the aptamer strategy to detect reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1). A direct and specific ACE method was proposed using laser-induced fluorescence (ACE/LIF) as detection principle. Single-stranded DNA aptamers as probes fluorescently labeled were synthesized. The resulting aptamer is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-l RT was stable, with calibration curves linear up to 50 nM (6 /xg/mL) HIV-1 RT concentration. Both... 

DNA in the test sample is denatured to give single-stranded DNA, immobilized on a nitrocellulose or other suitable filter and hybridized with labelled DNA prepared from the host-vector manufacturing system (DNA probes). Although a wide variety of experimental approaches is available, hybridization methods for measurement of host-vector DNA meet the following criteria ... 

Double-strand DNA is denatured to single-strand DNA. Exposed amines along DNA chains do not find reactive groups on the particle surface. 

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