Document Detail


Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection.
MedLine Citation:
PMID:  20307487     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments.
Authors:
Ekaterina Protozanova; Meng Zhang; Eric J White; Emilia T Mollova; Dirk Ten Broeck; Sergey V Fridrikh; Douglas B Cameron; Rudolf Gilmanshin
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2010-03-20
Journal Detail:
Title:  Analytical biochemistry     Volume:  402     ISSN:  1096-0309     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2010 Jul 
Date Detail:
Created Date:  2010-05-14     Completed Date:  2010-08-09     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  83-90     Citation Subset:  IM    
Copyright Information:
2010 Elsevier Inc. All rights reserved.
Affiliation:
U.S. Genomics, Woburn, MA 01801, USA.
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MeSH Terms
Descriptor/Qualifier:
Bacteria / genetics*
Bacterial Typing Techniques / methods
DNA Fingerprinting / methods*
DNA, Bacterial / genetics*
Escherichia coli / genetics
Genome, Bacterial*
Microfluidic Analytical Techniques / methods
Reproducibility of Results
Chemical
Reg. No./Substance:
0/DNA, Bacterial

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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