Nucleic Acids Research, 1989, Vol. 17, No. 22 9027-9037
© 1989
MOLECULAR BIOLOGY |
PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning
CRC Eukaryotic Molecular Genetics Research Group, Imperial College London SW7 2AZ and Cancer Research Campaign Laboratories, Department of Biochemistry, University of Dundee Dundee DD1 4HN, USA 1Department of Genetics, Cambridge University Downing Street, Cambridge, UK 2IMBB, Foundation of Research and Technology Heraklion, Crete, Greece 3Department of Cellular and Developmental Biology, Harvard University Cambridge, MA, USA
*To whom correspondence should be addressed
Received September 4, 1989. Accepted October 10, 1989.
A novel alternative to microcloning for the production of region specific chromosomal DNA is described. In this method, ‘microamplification’, single bands are dissected from polytene chromosomes and digested with Sau3A. Oligonucleotide adaptors are ligated to these fragments to provide convenient priming sites for polymerase chain reaction amplification. In this way, as much as 1µg of DNA can be amplified from a single band. Probes made from PCR amplified DNA from two such dissections have been used to probe cloned DNA form a 100kb chromosome walk. Whereas conventional microcloning has generated cloned EcoRI fragments corresponding to 3–4kb of the walk, the PCR probes cover greater than 90% of this chromosomal region. Thus microamplification is significantly more effective than microcloning in providing probes for establishing chromosomal walks.
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