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Anal Chem. 2011 Oct 1;83(19):7269-75. doi: 10.1021/ac2013465. Epub 2011 Aug 31.

Specific sorting of single bacterial cells with microfabricated fluorescence-activated cell sorting and tyramide signal amplification fluorescence in situ hybridization.

Author information

1
Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA. chc008@gmail.com

Abstract

When attempting to probe the genetic makeup of diverse bacterial communities that elude cell culturing, researchers face two primary challenges: isolation of rare bacteria from microbial samples and removal of contaminating cell-free DNA. We report a compact, low-cost, and high-performance microfabricated fluorescence-activated cell sorting (μFACS) technology in combination with a tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) to address these two challenges. The TSA-FISH protocol that was adapted for flow cytometry yields a 10-30-fold enhancement in fluorescence intensity over standard FISH methods. The μFACS technology, capable of enhancing its sensitivity by ~18 dB through signal processing, was able to enrich TSA-FISH-labeled E. coli cells by 223-fold. The μFACS technology was also used to remove contaminating cell-free DNA. After two rounds of sorting on E. coli mixed with λ-phage DNA (10 ng/μL), we demonstrated over 100,000-fold reduction in λ-DNA concentration. The integrated μFACS and TSA-FISH technologies provide a highly effective and low-cost solution for research on the genomic complexity of bacteria as well as single-cell genomic analysis of other sample types.

PMID:
21809842
DOI:
10.1021/ac2013465
[Indexed for MEDLINE]
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