DNA sequencing by hybridization: 100 bases read by a non-gel-based method.
Determination of the sequences of human and other complex genomes requires much faster and less expensive sequencing processes than the methods in use today. Sequencing by hybridization is potentially such a process. In this paper we present hybridization data sufficient to accurately read a known sequence of 100 base pairs. In independent reactions, octamer and nonamer oligonucleotides derived from the sequence hybridized more strongly to this DNA than to controls. The 93 consecutive overlapping probes were derived from a 100-base-pair segment of test DNA and additional probes were generated by incorporation of a noncomplementary base at one of the ends of 12 of the basic probes. These 12 additional probes also had a full-match target in one of the control DNAs. The test and one of five control DNAs spotted on nylon filters were hybridized with 83 octamers and 22 nonamers under low-temperature conditions. A stronger signal in DNA containing a full-match target compared to DNA with only mismatched targets was obtained with all 105 probes. In 3 cases (2.9%), the difference of signals was not significant (less than 2-fold) due to inefficient hybridization and the consequently higher influence of background. The hybridization pattern obtained enabled us to resequence the 100 base pairs by applying an algorithm that tolerates an error rate much higher than was observed in the experiment. With this result, the technological components of large-scale DNA sequencing using the sequencing by hybridization method are in place.
Strezoska
Z
Z
Institute of Molecular Genetics and Genetic Engineering, Belgrade, Yugoslavia.
Paunesku
T
T
Radosavljević
D
D
Labat
I
I
Drmanac
R
R
Crkvenjakov
R
R
D001483:Base Sequence; D002151:Calorimetry; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping
D015345:Oligonucleotide Probes; D007371:Interferon-gamma; D004247:DNA