Genomic signature tags (GSTs): a system for profiling genomic DNA.
Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. GSTs are shown to be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced to provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. GST analysis of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. The GST profile predicts that this strain has several changes relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity island.
Dunn
John J
JJ
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA. jdunn@bnl.gov
McCorkle
Sean R
SR
Praissman
Laura A
LA
Hind
Geoffrey
G
Van Der Lelie
Daniel
D
Bahou
Wadie F
WF
Gnatenko
Dmitri V
DV
Krause
Maureen K
MK
D001665:Binding Sites / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods*; D053938:DNA Fragmentation / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D015723:Gene Library; D016680:Genome, Bacterial; D008025:Ligases / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D015010:Yersinia pestis / Q000235:genetics
D004269:DNA, Bacterial; D009841:Oligonucleotides; D015245:Deoxyribonuclease BamHI; C049760:endodeoxyribonuclease MmeI; D015252:Deoxyribonucleases, Type II Site-Specific; D008025:Ligases