We will develop highly specific DNA cleavage tools, based on coupling adenine methylation with the methylation-dependent restriction-endonuclease Dpnl. We have produced DNA fragments of over 1,000,000 base pairs in size with one methylase/Dpnl cleavage system, and separated the resulting pieces by pulsed field gel electrophoresis. These results encourage us to develop the other methylase/Dpnl cleavage specificities based on known restriction modification systems, of which there are at least 22 possible. The cleavage specificities so generated recognize DNA sequences ranging from 8 to 14 base pairs in length and give average fragment sizes of 65,000 (4(8)) to 256,000,000 (4(14)) base pairs on random DNA. We intend to characterize, purify and clone those methylases that have the correct Gm6A specificity for use with Dpnl. This battery of methylase/Dpnl specificities can be applied to three mapping strategies: (1) The production of a physical map of the human genome using restriction fragment length polymorphism probes that have already been ordered genetically. (2) Construction of unique sites in insertion vectors such as retroviruses for the mapping of large portions of the human genome by Smith-Birnstiel indirect end-labelling. (3) Identification of 'linking clones' containing rare cleavage sites for the production of a physical map of these rare sites in the human genome.
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