Sequencing and Fingerprinting DNA by Hybridization with Oligonucleotide Probes
Nucleic Acids Structure and Mapping
Published Online: 15 SEP 2006
Copyright © 2000 John Wiley & Sons, Ltd. All rights reserved.
Encyclopedia of Analytical Chemistry
How to Cite
Drmanac, R., Drmanac, S. and Little, D. 2006. Sequencing and Fingerprinting DNA by Hybridization with Oligonucleotide Probes. Encyclopedia of Analytical Chemistry. .
- Published Online: 15 SEP 2006
Sequencing by hybridization (SBH) determines the sequences of DNA or RNA targets using hybridization data obtained from sets of overlapping oligonucleotide probes of known sequence. SBH shares interesting parallels with computer-based keyword searches of text files. In SBH, chemically synthesized short oligonucleotides (usually 4–25 bases in length) are hybridized to the target under conditions that predominantly allow formation of fully complementary probe/target hybrids. An oligonucleotide hybridization search is effectively a highly parallel molecular computation process with fully random access to the polynucleotide chain “input data”. The target sequence is assembled by aligning sequences of the subset of positively hybridizing oligonucleotides. Probe sequence overlap allows indirect assignment of which of four bases corresponds to each position in the analyzed DNA chain, without performing any actual positional measurements on the sample. The fundamental characteristics of the SBH process confer unique opportunities for miniaturization and parallel analyses (currently in the form of high-density arrays), leading to rapid, cost-effective DNA sequencing. A unique advantage of SBH is its ability to analyze in a single reaction complex DNA or RNA samples that may be thousands of bases long. Hybridization of smaller selected subsets of probes provides a very efficient way of partially sequencing and fingerprinting samples. A very important advantage of SBH is that in a single reaction each base is “read” by multiple overlapping probes. To achieve these advantages, hybridization reactions are miniaturized and coupled with automated data acquisition and efficient computational analyses that can process data sets containing both false-positive and false-negative probe scores. Successful assembly of long target sequences involves synthesis and hybridization of a large number of long probes, due to the higher probability that given short oligonucleotide sequences will be repeated within the target sequence. Overall, SBH is a rapid and cost-effective method of obtaining large amounts of accurate DNA sequence information.