Randomized oligonucleotides

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. 

Owens, G.C., Chappell, S.A., Mauro, V.P. and Edelman, G.M. (2001) Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides. Proc. Natl. Acad. Sci. USA, 98,1471-1476. 

Peng, L., Stephens, B.J., Bonin, K. et al. 2007b. A combined atomic force/fluorescence microscopy technique to select aptamers in a single cycle from a small pool of random oligonucleotides. Microsc Res Tech 70(4) 372-381. 

Another way to design randomized oligonucleotides is to synthesize triplets as NNY and RNN, where Y represents dC or T and R represents dA or dG [33], all in equimolar mixtures. This design eliminates stop codons, but does not contain two of the 20 amino acids, and hence limiting the diversity. 

We describe the various approaches that one might use to generate novel enzymes and the application of one such technique, Random Oligonucleotide Mutagenesis, to the DNA synthesis and repair enzymes DNA polymerase I from E. coli and Thermus aquaticus, as well as human thymidylate synthase, thymidine kinase and 06-alkylgua-nine-DNA alkyltransferase. 

Site Directed Chemical /X-Rny FCK. Mutagenesis DNA Shuffling Random Oligonucleotides... 

Random oligonucleotide mutagenesis was first applied to promotor sequences that regulate the production of enzymes in cells [21] and was the first method used to alter systematically the functions of enzymes by directed evolution [22], Based on our experience, we will focus on this approach and emphasize recent applications of this methodology to enzymes involved in DNA repair and synthesis, including DNA polymerase enzymes, thymidylate synthase, thymidine kinase, and 06-alkylguanine-DNA alkyltransferase. 

The DNA to be radiolabeled (0.1 -0.3 pg) is mixed with a molar excess of random oligonucleotides and denatured in a boiling water bath for 1-3 min, and... 

An alternative approach to the synthesis of random oligonucleotides which code for different proteins has been described. Trinucleotide phosphoramidites 134 coding for all 20 amino acids have been synthesised and utilised to assemble oligodeoxyribonucleotide libraries. 

In a series of well-controlled studies, anti-sense oligonucleotides administered intraven-tricularly selectively inhibited dopamine type 2 receptor expression, dopamine type 2 receptor RNA levels, and behavioral effects in animals with chemical lesions. Controls included randomized oligonucleotides and the observation that no effects were observed on dopamine type I receptor or RNA levels (142-144). This laboratory also reported the selective reduction of dopamine type 1 receptor and RNA levels with the appropriate oligonucleotide (227). 

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