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| SEQUENCING BY HYBRIDIZATION - TOWARDS AN AUTOMATED SEQUENCING OF ONE MILLION M13 CLONES ARRAYED ON MEMBRANES |
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| Author(s): DRMANAC R, DRMANAC S, LABAT I, CRKVENJAKOV R, VICENTIC A, GEMMELL A |
| Source: ELECTROPHORESIS Volume: 13 Issue: 8 Pages: 566-573 Published: AUG 1992 |
| Times Cited: 32 References: 34 |
| 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 M 13 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 P-32-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. |
| Document Type: Article |
| Language: English |
| Reprint Address: DRMANAC, R (reprint author), ARGONNE NATL LAB, DIV BIOL & MED RES, 9700 S CASS AVE, ARGONNE, IL 60439 USA |
| Publisher: VCH PUBLISHERS INC, 303 NW 12TH AVE, DEERFIELD BEACH, FL 33442-1788 |
| Subject Category: Biochemical Research Methods; Chemistry, Analytical |
| IDS Number: JP282 |
| ISSN: 0173-0835 |
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