Rapid Preparation of DNA for Automated Sequencing 



John J. Dunn and F. William Studier 



Biology Department, Brookhaven National Laboratory, Upton, NY 1 1973 



(316)282-3012, FTS 666-3012 



A strategy that uses a library of oligonucleotide primers of length eight, nine, or ten has 

 been developed for direct sequencing of cosmid DN As. The statistics of priming 

 indicate that a library sufficient for determining the sequence of hundreds of thousands 

 of different cosmids could be readily assembled. This strategy would greatly reduce the 

 cost and effort of human genome sequencing. Any needed primer would be instantly 

 available at a cost of considerably less than 0.1 cent per nucleotide of sequence 

 obtained. Mapping, subcloning, or preparation of multiple DNA samples would not be 

 necessary, and the wasteful redundancies of random sequencing strategies would be 

 eliminated. The success of this strategy requires only that a considerable fraction of all 

 octamers, nonamers, or decamers be able to prime selectively. Work is under way to 

 establish conditions where this will be the case. The transposon gamma-delta is being 

 modified to carry genetic signals that enable bacteriophage T7 to replicate and package 

 plasmid DNAs. The ability of such an element to insert these signals into a cosmid 

 DNA and thereby to facilitate preparation of the DNA for sequencing is being tested 

 using a cosmid that carries a complete genomic copy of the receptor gene for polio 

 virus. 



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