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Content archived on 2024-04-16

Development of high speed DNA sequencing technology for sequencing of human cDNAs and microsatellite DNA


The objective and result of this collaboration among European scientists will be the development and improvement of advanced technologies which will facilitate the physical mapping of the human genome.
Basic physical parameters for the high speed deoxyribonucleic acid (DNA) sequencer have been identified and a continuous detector array for simultaneous DNA sequencing of 20 clones on the standard gel formation has been developed.
The development of the 2-dye sequencing system has been completed. Based on the previous sequencer a novel strategy has been developed allowing to sequence on both strands of a double stranded template simultaneously in the same sequencing reaction, and to detect both sequences online.
New detectors have been designed for the collection of the fluorescent light emitted by the DNA samples labelled with 2 different fluorophores, allowing 8 samples per each fluorophore.
a technique with vibrating elements shows the greatest promise for automated casting.
Existing methodology developed for the preparation of M13 single stranded DNA was modified and tested for its applicability for the purification of double stranded DNA. Different membrane materials were tested with respect to their DNA binding ability and capability.
About 600 randomly picked chromosomal deoxyribonucleic acid (cDNA) clones from a normal keratinocyte cDNA library have been picked and sequenced from the ends. So far 24 full length clones were recombined into the vaccinia virus system and expressed the proteins in humans antimitochondrial antibody (AMA) cells. This allowed the mapping of these proteins to spots in the 2-dimensional gel database.
More than 10 full length unknown cDNA clones have been completely sequenced, additional 20 cDNA clones have been partially sequenced. These partial cDNAS were used as probes for screening cDNA libraries, and the corresponding full length clones have been isolated.
The sequencing was completed using the waling primer strategy, and labelled deoxynucleotide triphosphates (dNTPs). Within 3 bases following the primer a labelled dNTP has to be incorporated in order to ensure efficient labelling of the DNA strands.
A total of 190 000 bases of sequence have been collected and 154 human polymorphic microsatellite clones have been analyzed.
To characterize fully parts of the human genome means to determine the complete sequence of those parts. Recent physical mapping techniques involve the determination of short stretches of sequence spaced at certain intervals along the genome (STS mapping).

The automated methods currently available, based on polyacrylamide technology and fluorescent labelling of DNA fragments, allow a sequence output of about 10 kilo bases per device per day. Characterization of the genomic regions of interest would take a prohibitively long time with these techniques. Systems allowing 10 to 20 times higher sequence outputs (100 to 200 kilo bases per device per day) are required as an efficient tool for sequence characterization of genomes.

Such high speed sequencing systems could be constructed, based upon ultrathin (about 100 microns thickness) polyacrylamide gel technology in combination with fluorescently labelled DNA fragments, development of new, 5 to 10 times more sensitive detectors of fluorescent signals, allowing sequencing speeds of 1000 to 2000 bases per hour.

The daily sequence output from the same number of gels will be further doubled by development of a sequencing system (based upon the EMBL design) using two fluorescent dyes simultaneously. This high speed, high output technology will be applied to the sequencing and mapping of cDNAs and of microsatellite DNA.

With the high sequencing speed and short duration of a sequencing run on one gel, the development of techniques enabling reloading of the same gel several times becomes an important operational feature. For the same reasons, to assure high sequence output, an automated system for loading of samples on the ultrathin gels will be developed. Human cDNAs derived from cell type specific 2-dimensional protein gels will be sequenced and STS mapped. A direct link between a protein, its coding nucleotide sequence and physical localization on the genome will be established and this information integrated into existing databases.

Using the technology described above, polymorphic simple repeat microsatellite DNA sequences from human chromosomes 9 and 21 will be generated by PCR or hybridization techniques and sequenced.


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