A microarray-based method to perform nucleic acid selections.
This method describes a microarray-based platform to perform nucleic acid selections. Chemical ligands to which a nucleic acid binder is desired are immobilized onto an agarose microarray surface; the array is then incubated with an RNA library. Bound RNA library members are harvested directly from the array surface via gel excision at the position on the array where a ligand was immobilized. The RNA is then amplified via RT-PCR, cloned, and sequenced. This method has the following advantages over traditional resin-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX): (1) multiple selections can be completed in parallel on a single microarray surface; (2) kinetic biases in the selections are mitigated since all RNA binders are harvested from an array via gel excision; (3) the amount of chemical ligand needed to perform a selection is minimized; (4) selections do not require expensive resins or equipment; and (5) the matrix used for selections is inexpensive and easy to prepare. Although this protocol was demonstrated for RNA selections, it should be applicable for any nucleic acid selection.
Aminova
Olga
O
Department of Chemistry and The Center for Excellence in Bioinformatics and Life Sciences, University at Buffalo, Buffalo, NY, USA.
Disney
Matthew D
MD
D000588:Amines / Q000737:chemistry; D000617:Aminoglycosides / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010084:Oxidation-Reduction; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose / Q000737:chemistry; D013499:Surface Properties; D014158:Transcription, Genetic
D000588:Amines; D000617:Aminoglycosides; D008024:Ligands; D009696:Nucleic Acids; D012313:RNA; D004247:DNA; D012685:Sepharose