Nanoliter reactors improve multiple displacement amplification of genomes from single cells.
Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.
Marcy
Yann
Y
Department of Bioengineering, Stanford University, Stanford, California, USA.
Ishoey
Thomas
T
Lasken
Roger S
RS
Stockwell
Timothy B
TB
Walenz
Brian P
BP
Halpern
Aaron L
AL
Beeson
Karen Y
KY
Goldberg
Susanne M D
SM
Quake
Stephen R
SR
D005784:Gene Amplification; D016678:Genome; D017404:In Situ Hybridization, Fluorescence; D044085:Microfluidics; D036103:Nanotechnology; D015347:RNA Probes
D015347:RNA Probes