Selection of oligonucleotide probes and experimental conditions for multiplex hybridization experiments.
Different DNA probes hybridize under different conditions. I examine the constraints of the design of oligonucleotide probes that are meant to hybridize to different unique sites in human genomic DNA under a single set of hybridization conditions as a parallel array. In 522 kb of human genomic DNA, 75% of 12-base and 89% of 22-base are unique, as opposed to 90% and 100% as expected of unstructured DNA, and this is not due solely to repetitive elements in the DNA. Hybridization in TMAC to reduce A+T content effects on melting temperature allows only 90% of unique targets to be hybridized under one set of conditions if a 2 degrees C difference between matched and mismatched sequences is required. Standard hybridization conditions allow no more than 60% of unique probes to be used together. This suggests that probe, hybridization conditions, and instrument design for multiple-probe hybridization applications will be harder than previously suggested.
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PA Consulting Group, Royston, Herts, England.
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013696:Temperature
D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes; D015345:Oligonucleotide Probes; D004247:DNA