Sequencing by hybridization: Towards an automated sequencing of one million M13 clones arrayed on membranes

Abstract

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864-well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96-well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13,824 samples on an 8 x 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200-2000 32P-labeled probes comprising 16-256 10-mers having a common 8-mer, 7-mer, or 6-mer in the middle (20 probes per day, each hybridized with 250,000 dots); (v) scoring hybridization signals of 5 million sample-probe pairs per day using storage phosphor plates; and (vi) computing clone order and partial-to-complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

“…Most of the DNA hybridization detection methods employed thus far use radioactive (15)(16)(17)(18)(19)(20), enzyme-based chemiluminescent (21), or fluorescent (22) labels. Detection and measurement can be accomplished with phosphor systems (23) or, for the latter two labels, with charge-coupled device (CCD) cameras (24), cooled CCD cameras, image intensifiers coupled to CCD cameras, or a photomultiplier tube coupled with mechanical raster scanning (12,25).…”

“…AF508 indicates a 3-bp deletion that results in removal of Phe-508 of the cystic fibrosis transmembrane conductance regulator polypeptide (31). The G -* A change at codon 551 results in a change from Gly to Asp (G551D) and the C --T change results in a stop codon in place of the normal codon for Arg (R553X) (16). WT indicates the wild-type or normal sequence at each position.…”

“…While not physiologically relevant, the double mutation was included to evaluate chip performance. Complementary oligonucleotides [11][12][13][14][15][16][17][18][19] for 32P-end-labeling and oligonucleotides 21B-29B with 3'-biotin labels for wave guide detection were synthesized. Fig.…”

Discovering distinct genes represented in 29,570 clones from infant brain cDNA libraries by applying sequencing by hybridization methodology.

“…Clones from the original and normalized human infant brain cDNA libraries (Soares et al 1994) were arrayed and immobilized on nylon filters in the form of PCR products of plasmid inserts by applying previously described techniques (Drmanac and Drmanac 1994;Drmanac et al 1992Drmanac et al , 1993Grujic et al 1994).…”

“…The hybridization data production lines that are being developed for the purpose of sequencing by hybridization (SBH) can be employed easily for rapid generation of OSSs (Drmanac et al 1992(Drmanac et al , 1993Meier-Ewert et al 1993;Drmanac and Drmanac 1994;Grujic et al 1994). Densely arrayed clones can be examined at a 10-to 100-fold higher rate and much more economically than by standard sequencing, thus achieving the data throughputs required for exhaustive gene discovery and for detailed studies of expression patterns.…”