Sequence scanning: A method for rapid sequence acquisition from large-fragment DNA clones.
A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb. A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers. Then long-range spectrum PCR is used to order and orient the clones. This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes. Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps. Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence.
Nurminsky
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Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA.
Hartl
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D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant / Q000737:chemistry*; D004330:Drosophila / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
D004274:DNA, Recombinant