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Effect of Oligonucleotide Truncation on Single-Nucleotide Distinction by Solid-Phase Hybridization

2001; American Chemical Society; Volume: 74; Issue: 1 Linguagem: Inglês

10.1021/ac010555s

1520-6882

Magnus Jobs, Simon Fredriksson, Anthony J. Brookes, Ulf Landegren,

Advanced Biosensing Techniques and Applications

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.