Creation of ultra-rare restriction sites in intact eucaryotic chromosomes mediated by bacterial methylases: an approach to sequencing and analyzing tumor and normal genomes.
The limited restriction of eucaryotic chromosomes would facilitate our understanding of the aberrant genomes of genetic diseases and cancer. We have described methods for methylating eucaryotic chromosomes embedded in agarose plugs. We now describe how Cla I methylase can be utilized in a methylation-dependent restriction cleavage with Dpn I to restrict eucaryotic genomes into a limited number of fragments. We have restricted the genomes of Saccharomyces cerevisiae at four sites and cleaved the three chromosomes of Schizosaccharomyces pombe into eleven fragments. Unlike the recently published methodologies using DNA sequences inserted into procaryotic and eucaryotic genomes, the methodologies described here use unaltered eucaryotic genomes for highly limited restriction. This methodology has applications in speeding, simplifying, and reducing the cost of sequencing the human genome.
Wilson
W W
WW
Laboratory of Cancer Biology, University of California, San Diego, La Jolla 92093-0609.
Mebane
E W
EW
Hoffman
R M
RM
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field; D016681:Genome, Fungal; D008745:Methylation; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D001426:Bacterial Proteins; D004271:DNA, Fungal; D004273:DNA, Neoplasm; C020102:endodeoxyribonuclease DpnI; C032824:ATCGAT-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific