Genome mapping by fluorescent fingerprinting.
The construction of sequence-ready maps of overlapping genomic clones is central to large-scale genome sequencing. We have implemented a method for fluorescent fingerprinting of bacterial clones to assemble contig maps. The method utilizes three spectrally distinct fluorescently tagged dideoxy ATPs to specifically label the HindIII termini in HindIII and Sau3AI restriction digests of clones that are multiplexed prior to electrophoresis and data collection. There is excellent reproducibility of raw data, improved resolution of large fragments, and concordance between the results obtained using this and the equivalent radioactive protocol. This method also allows detection of smaller overlaps between clones when compared to the analysis of restriction digests on nondenaturing agarose gels.
Gregory
S G
SG
The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. sgg@sanger.ac.uk
Howell
G R
GR
Bentley
D R
DR
D002874:Chromosome Mapping / Q000379:methods*; D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D054306:Dideoxynucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism
D004269:DNA, Bacterial; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; C037773:2',3'-dideoxyadenosine triphosphate; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases