Random oligonucleotide

By similar logic, protein affinity Hbraries have been constmcted to identify protein—protein combining sites, as in antibody—antigen interaction (19) and recombinant Hbraries have been made which produce a repertoire of antibodies in E. coli (20). In another case, a potential DNA-based therapeutic strategy has been studied (21). DNAs from a partially randomized Hbrary were selected to bind thrombin in vitro. Oligonucleotides, termed aptamers that bound thrombin shared a conserved sequence 14—17 nucleotides long. 

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

Differential display is a method for identifying differentially expressed genes, using anchored oligo-dT, random oligonucleotide primers and polymerase chain reaction on reverse-transcribed RNA from different cell populations. The amplified complementary DNAs are displayed and comparisons are drawn between the different cell populations. 

The unique properties of oligonucleotides create crosslinking options that are far different from any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically duplicated in vitro by enzymatic means. The addition of modified nucleoside triphosphates to an existing DNA strand by the action of polymerases or transferases allows addition of spacer arms or detection components at random or discrete sites along the chain. Alternatively, chemical methods that modify nucleotides at selected functional groups can be used to produce spacer arm derivatives or activated intermediates for subsequent coupling to other molecules. 

Relatively short RNA oligonucleotides were first formed by random events. 

Cleavage in the presence of histidine releases the oligonucleotide fragment containing the random sequence, and this is amplified by PCR and the cycle repeated. This selection procedure produced deoxyribozymes that require no metal cofactor but have a specific requirement for L-histidine, which is presumed (from pH-rate profiles) to act as a general... 

Illumina produces fiberoptic random bead arrays. Latex beads are encoded using different fluorescent dye mixtures that are either adsorbed into the particles or attached to the surfaces. Presynthesized oligonucleotides are attached to selected bead populations so that a single dye or dyerdye ratio identifies the attached oligonucleotide. Populations are mixed in bulk and then loaded onto the tips of a fiberoptic, one end of which has been acid etched to form microscopic nanowells. The nanowells are filled at random with the mixed bead population to create a BeadArray . Such bxmdles can... 

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