Representative and efficient cloning of satellite DNAs based on PFGE pre-fractionation of restriction digests of genomic DNA.
Using DNA from Drosophila hydei KUN-DH-33 cells we describe an efficient method for selective and representative cloning of complex mixtures of satellite DNAs from eukaryotic genomes. Effective separation of satellite DNA from the bulk of all other sequences it obtained by fractionation of high molecular weight DNA by PFGE after treating it with '6 bp' restriction enzymes. Since extended clusters of tandemly arranged, so called simple sequence, repeats are inert to cleavage by most '6 bp' restriction enzymes the DNA fraction recovered from the gel region > 50 kb is mainly a mixture of satellites. Efficient and representative cloning of this DNA is performed by sonication to an average size of 50-500 bp and ligation of the blunt ended DNA fragments into the Bluescript vector pBS.
Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, cermany.
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Burgtorf
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Bünemann
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005591:Chemical Fractionation; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes; D004276:DNA, Satellite / Q000235:genetics*; D004330:Drosophila / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D006882:Hydroxyapatites; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012091:Repetitive Sequences, Nucleic Acid; D014998:Y Chromosome
D004276:DNA, Satellite; D006882:Hydroxyapatites; D004262:DNA Restriction Enzymes