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. 2005 Jun;71(6):3342-7.
doi: 10.1128/AEM.71.6.3342-3347.2005.

Genomic DNA amplification from a single bacterium

Arumugham Raghunathan  1 , Harley R Ferguson Jr, Carole J Bornarth, Wanmin Song, Mark Driscoll, Roger S Lasken
Affiliations

Genomic DNA amplification from a single bacterium

Arumugham Raghunathan et al. Appl Environ Microbiol. 2005 Jun.

Abstract

Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using phi 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying non-culturable microorganisms.

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Figures

FIG. 1.
FIG. 1.
Verification of single-cell isolation by flow sorting. (A) Two examples of the five plates (42 squares per plate grid). (B) Colonies growing from each well were counted after an overnight incubation.
FIG. 2.
FIG. 2.
DNA sequencing of the 16S rRNA gene by direct primer annealing to DNA amplified from a single cell. Five microliters of DNA amplified from a single cell was sequenced on an Applied Biosystems 3730 capillary DNA analyzer.
FIG. 3.
FIG. 3.
Analysis of amplified DNA by quantitative PCR. (A) Each bar is the average of results for four different loci and 10 replicate MDA reactions for approximately 100, 50, and 10 flow-sorted E. coli cells. Error bars are 1 standard deviation. (B) Single cells were sorted into 84 microtiter plate wells, lysed, and subjected to whole-genome amplification by MDA. Average locus representation (n = 84) is plotted on the y axis for 10 individual loci in the E. coli genome. The average of all 10 loci is represented in the last bar. (C) Amplified DNA (negative controls and single-cell samples) resolved by agarose gel electrophoresis. Lanes M, molecular size markers.
FIG. 4.
FIG. 4.
Whole-genome amplification from 1 or 10 M. xanthus cells. Results of a quantitative assay for a representative locus are shown.
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