Truncation SELEX method
a selex method and truncation technology, applied in the field of identifying oligonucleotide sequences, can solve the problems of limiting the number of different nucleic acid ligands and the possibility of structural variation
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example 1
Experimental Procedures for Performing the SELEX Process by Annealing of the Complementary Oligonucleotides to the Fixed Sequence Region or by Changing the Fixed Sequences
[0125] This example provides general procedures followed by and incorporated into Example 2.
Materials and Methods
[0126] The library from E. coli B genomic DNA was constructed as described previously (Singer et al. (1997) Nuc. Acids Res. 25:781-786). Briefly, genomic DNA was denatured and annealed to a primer with a fixed 5′ end and 9 randomized nucleotides at the 3′ end. After annealing at 2° C., the primer was extended with Klenow on ice, followed by room temperature, and 50° C. Another primer with a different fixed sequence was added, and the denaturation / annealing / extension was repeated. The molecules were separated by size on a denaturing polyacrylamide gel, and amplified by PCR using the above two primers minus the randomized sequences, plus the T7 promoter.
Genomic SELEX
[0127] A non-agg...
example 2
Binding Sites from the MS2 CP SELEX Agree with the Known Consensus Structure
[0155] A library of genomic DNA was prepared from E. coli B by random primer extension (Singer et al. (1997) Nucleic Acids Research 25:781-786). The library contained approximately 65 nucleotide genomic inserts flanked by fixed sequences, which serve as primer annealing sites for amplification. Insert refers to the genomic sequence located in the library molecule between the two fixed sequences. In each round of SELEX, the transcribed library was allowed to bind MS2 CP, and then the bound RNA was amplified. In SELEX experiment 1, the DNA was cloned and sequenced after 5 rounds, when the optimal binding was observed.
[0156] Out of 25 isolates sequenced, 12 had the predicted consensus (Witherell et al. (1991) Prog Nucleic Acid Res. Mol. Biol. 40:185-220) binding site (FIG. 2A), which could be identified either by folding by hand, or by computerized Zuker-Turner folding (Genetics Computer Group, Program Manua...
example 3
[0183] This Example provides general procedures followed by and incorporated into Examples 4-12.
Materials
[0184] Recombinant human Transforming Growth Factor Beta 1 (hTGFβ1) and human Vascular Endothelial Growth Factor (VEGF) were from R&D Systems (Minneapolis, Minn.). DNA and RNA modifying enzymes were from Roche Molecular Biochemicals (Indianapolis, Ind.), BRL (Gaithersburg, Md.), or NEB (Beverly, Mass.) or PE (Foster City. Calif.). Biotin-21-dUTP was from Clontech (Palo Alto, Calif.), terminal deoxynucleotidyl transferase was from Clontech (Palo Alto, Calif.) or Roche Molecular Biochemicals (Indianapolis, Ind.). T7 RNA polymerase, 2′F-modified CTP and UTP were prepared in house. Taq DNA polymerase was from Perkin Elmer (Foster City, Calif.). DNA oligonucleotides were obtained from Operon Technologies, Inc. (Alameda, Calif.). All other reagents and chemicals were from commercial sources.
Affinity Selection (SELEX)
[0185] The SELEX procedure has been described in detail in the S...
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