Solid Phase Hybridization Techniques

Peptide nucleic acids can be used in many of the same hybridization applications as natural or synthetic DNA probes but with the added advantages of tighter binding and higher specificity. This leads to faster and easier procedures in most standard hybridization techniques, such as Southern and Northern blotting. An alternative to Southern analysis is also the PNA pregel hybridization process, which significantly simplifies the procedure of Southern hybridization.[15] Labeled PNAs are then used as probes, allowing hybridization to a denatured dsDNA sample at low ionic strength before loading on the gel. The method is sensitive enough to detect a single mismatch in a DNA sample. Another gel-based strategy, known as affinity electrophoresis can be exploited with PNA probes. In this case, the slowdown of DNA during electrophoretic passage is related to its hybridization with a complementary PNA probe entrapped in the gel matrix. Likewise, hybridization PNA-based biosensor procedures have been developed in which a single-stranded PNA probe is immobilized onto optical or mass-sensitive transducers to detect the complementary strand or corresponding mismatch in a DNA sample solution. The hybridization events are converted into electric signals by the transducers.1-16-1

(FISH) applications. The properties of PNAs have allowed the development of fast, simple, and robust in situ assays. Because of their neutral backbone, PNA probes present in situ high specificity and require low concentrations and short hybridization time. Additional in situ benefits of using PNA probes are reduced background binding, low photobleaching, and mild washing procedure. In situ labeling can efficiently be obtained with a single 15-mer PNA oligomer carrying a single label.

The PNA-FISH technique was first developed for quantitative telomere analysis. Using a unique fluoresce-in-labeled PNA probe, Lansdorp et al.[17] performed the in situ labeling of human telomeric repeat sequences and the data obtained allowed accurate estimates of telomere lengths. Subsequently, telomeric PNA probes were used in several in situ studies of cancer and aging.[18] Further developments were focused on the in situ specific identification of human chromosomes on both metaphases and interphase nuclei, using PNA probes specific for satellite repeat sequences of various chromosomes. Multicolor PNA experiments were thus reported on lymphocytes (Fig. 2), amniocytes, and fibroblasts from normal subjects and patients with numerical abnormalities.1-19,20-1 These experiments demonstrated the superiority of PNA probes over satellite DNA probes in both intensity and sequence discrimination. Chen et al.[21] reported that PNA probes could discriminate in situ between two centromeric DNA repeats that differ by only a single base pair. The discriminating power of PNA could be very useful for the study of chromosomal variations and

Fig. 2 In situ identification of chromosomes 1, X, and Y on human metaphase and interphase nuclei using multicolor PNA-FISH procedure with satellite-specific PNA probes. Chromosome 1 is labeled in blue, chromosome X in red, and chromosome Y in green. (View this art in color at www.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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