Strand-specific DNA probes

Strand-specific DNA probes of high specific radioactivity are prepared by first annealing a primer to the single-stranded M13 DNA and then extending it with DNA polymerase (34). Annealing is accomplished in the presence of 100-fold molar excess of primer to DNA template (0.02-2 pg) in a reaction mixmre containing 50 mM NaCl, lOmMTris HCl, pH 7.5,and 7 mM MgCl2. The mixture is... 

For some applications, RNA probes are desirable because RNA-DNA hybrids are thermodynamically more stable than DNA-DNA hybrids. In addition, while the method described for production of strand-specific DNA probes requires purification of the radiolabeled DNA away from the unlabeled template, radioactive RNA probes may be obtained simply by treatment of the reaction mixture with RNase-free DNase I following RNA synthesis. 

An EMSAs is a simple assay to determine if a DNA sequence is able to bind a specific protein. Purified, cellular, or nuclear protein extract is incubated with labeled double-stranded oligonucleotide DNA probes, usually 20-25 nucleotides long. Each probe contains the SNP nucleotide (one per probe) and the identical sequence corresponding to the sequence around the SNP. The mixture is then run on a nondenaturing gel. If the DNA sequence binds the protein, there is a shift in the mobility of the complex (e.g., lane 2, Fig. 12.3). Specific and nonspecific competitors are often added to demonstrate specificity of the shift (lanes 5-7, Fig. 12.3). If an antibody... 

Fig. 5. Gmhspl7.6-L antisense RNA in transgenic tobacco and levels of endogenous hs mRNAs. Sense (C) and antisense constructs (D) as depicted in Fig. 3 were used for transformation of tobacco. Total RNA was isolated from leaves of individual plants which had been subjected to heat stress (hs) for 2 h at 40 °C, or incubated at 25 °C (c). 30 pg RNA per lane were separated by electrophoresis and blots were hybridised (A) with the same gene-specific DNA probe as used in Fig. 4. This hybridisation identifies the levels of antisense RNA in individual plants Dj, D2 and D3. Hybridisation (B) of an identical blot with a single stranded antisense RNA probe representing only the inverted region of construct D (Fig. 3), shows the levels of hs-induced endogenous tobacco hsp-mRNAs. tob, untransformed tobacco plants used as a control.

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). 

Koch, J., Hindkjaer, J., Mogensen, J., Kolvraa, S., and Bolund, L. (1991) An improved method for chromosome-specific labeling of alpha satellite DNA in situ using denatured double stranded DNA probes as primers in a PRimed IN Situ labeling (PRINS) procedure. GATA 8(6), 171-178. 

Chemical and Genetic Probes—Nanotube-tipped atomic force microscopes can trace a strand of DNA and identify chemical markers that reveal DNA fine structure. A miniaturized sensor has been constructed based on coupling the electronic properties of nanotubes with the specific recognition properties of immobilized biomolecules by attaching organic molecules handles to these tubular nanostructures. In one study, the pi-electron network on the CNT is used to anchor a molecule that irreversibly adsorbs to the surface of the SWNT. The anchored molecules have a tail to which proteins, or a variety of other... 

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