nanoliter reactors improve multiple displacement amplification of genomes from single cells只反应堆提高多个位移放大单个细胞的基因组.pdfVIP

Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells 1,2¤ 3 3 4 4 4 Yann Marcy , Thomas Ishoey , Roger S. Lasken , Timothy B. Stockwell , Brian P. Walenz , Aaron L. Halpern , 4 5 1,2* Karen Y. Beeson , Susanne M. D. Goldberg , Stephen R. Quake 1 Department of Bioengineering, Stanford University, Stanford, California, United States of America, 2 Howard Hughes Medical Institute, Stanford University, Stanford, California, United States of America, 3 J. Craig Venter Institute, La Jolla, California, United States of America, 4 J. Craig Venter Institute, Rockville, Maryland, United States of America, 5 The Joint Technology Center, J. Craig Venter Institute, Rockville, Maryland, United States of America 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-ll volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. S