Oligonucleotide probe-based

Homology screening. Using oligonucleotide probes based on known receptor sequences, search cDNA libraries for homologous sequences which may code for related receptors. The clones are then isolated and sequenced and used in expression studies to confirm the identity of the receptor. 

With the use of oligonucleotide probes based on the amino acid sequences of these protease V8-obtained peptides and of cyanogen bromide fragments of the porcine H,K-ATPase P subunit, cDNA clones for the rat [12,25] and rabbit [74] H,K-ATPase P subunit were then isolated. 

Antibodies can also be used as probes to screen expression libraries, and this strategy has been used successfully in the cloning of vertebrate neuropeptides (17). It is likely that this technique will be helpful in cases in which structural data is particularly difficult to obtain and a variety of highly specific antibodies is available. While these and other techniques will prove valuable in special circumstances, screening with synthetic oligonucleotide probes based on protein data is likely to be the principal method of identifying other peptide precursors in the near future. 

The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. Using oligonucleotide probes based on amino-terminal sequence data, the corresponding human gene from a genomic DNA library, has been cloned and expressed in animal cells. The protein is secreted from cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known PLAjS, and has a half-cysteine pattern that is characteristic of the snake venom group II enzyme (60). 

General Caveats of Oligonucleotide Probe-based Identification... 

Detection of an expressing clone was attempted by the use of a radioactive oligonucleotide probe based on the iV-terminal amino acid sequence. The probes used are shown in Fig. 4. However, this approach was not successful due to the degeneracy of the sequence. This approach may also identify nonexpressing forms of the lactamase such as truncated versions of the gene, and a direct screen for activity is more desirable and, in this case, necessary. 

Figure 1.57 Base-pairing can occur between complementary bases in opposing oligonucleotide strands. These predictable interactions form the basis for using synthetic oligonucleotide probes to target particular DNA sequences.

To modify the unique chemical groups on nucleic acids, novel methods have been developed that allow derivatization through discrete sites on the available bases, sugars, or phosphate groups (see Chapter 1, Section 3 for a discussion of RNA and DNA structure). These chemical methods can be used to add a functional group or a label to an individual nucleotide or to one or more sites in oligonucleotide probes or full-sized DNA or RNA polymers. 

When photobiotin is irradiated in the presence of DNA the reaction process nonselectively couples a biotin label to every 100-200 base residues. The result is an oligonucleotide probe detectable by the use of (strept)avidin conjugates. The uses of photobiotin for DNA or RNA modification are summarized in Chapter 11, Section 4. 

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