Combinatorial decoding: an approach for universal DNA array fabrication.
A fiber optic microsphere-based oligonucleotide array is described that employs the sequence of the oligonucleotide probe attached to each microsphere as positional identifiers. Each microsphere serves as an immobilized array feature, functionalized with a unique single-stranded oligonucleotide sequence and randomly distributed into an array of microwells. To determine the sequences attached to individual microspheres, a series of fluorescently labeled combinatorial-pooled oligonucleotide target solutions was designed. Each combinatorial decoding solution is intended to identify the nucleotide at a particular position on every microsphere in the array. The combinatorial target solutions were synthesized by linking the four possible nucleotides at each position to four different fluorescent reporter dyes. As such, when the solutions were hybridized to the array, one of four possible fluorescent responses was generated for each position on a microsphere probe sequence. Adjusting the stringency of hybridization enabled single-base mismatch discrimination, and the signal with the highest intensity corresponded to the perfect nucleotide match. By consecutively exposing the array to a series of combinatorial decoding pool solutions, it was possible to simultaneously determine the sequence of every randomly positioned oligonucleotide-functionalized microsphere in the array. Once mapped, the microsphere array can be used for any typical genomic microarray experiment.
Epstein
Jason R
JR
Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, Massachusetts 02155, USA.
Ferguson
Jane A
JA
Lee
Kyong-Hoon
KH
Walt
David R
DR
D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics; D005336:Fiber Optic Technology / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D055100:Optical Fibers
D015342:DNA Probes; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes