Effect of oligonucleotide truncation on single-nucleotide distinction by solid-phase hybridization.
Oligonucleotide microarrays are used to analyze target sequences on the basis of differences in hybridization stability between matched and mismatched probe-target duplexes. DNA microarray manufacture via photolithographic synthesis generates a minority of full-length oligonucleotide probes along with a series of 5'-truncated contaminants. In a model experiment, we now investigate the effect of truncated oligonucleotides on the ability to distinguish target sequence variants that differ in a single nucleotide position. A series of oligonucleotides, mixed in proportions simulating stepwise synthetic yields of between 82 and 100%, were bound to a solid support and allowed to hybridize to a target molecule. The extent of hybridization was monitored over a range of temperatures via the fluorescence of a double-strand-specific dye. The discriminatory power of pure oligonucleotide probes was found to be significantly greater than that of a population of truncated probes, but only over a limited temperature interval. We conclude that at optimal temperatures greater oligonucleotide quality can improve the performance of oligonucleotide hybridization microarrays.
Jobs
Magnus
M
Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, Sweden.
Fredriksson
Simon
S
Brookes
Anthony J
AJ
Landegren
Ulf
U
D000818:Animals; D015342:DNA Probes / Q000737:chemistry / Q000592:standards*; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards*; D009841:Oligonucleotides / Q000737:chemistry / Q000592:standards
D015342:DNA Probes; D009841:Oligonucleotides