Linked Life Data

18410131

Source:http://linkedlifedata.com/resource/pubmed/id/18410131

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2008-5-15
pubmed:abstractText
A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
1520-6882
pubmed:author
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
80
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3522-9
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets.
pubmed:affiliation
Department of Mechanical Engineering, UCSF/UC Berkeley Joint Bioengineering Graduate Group, University of California, Berkeley, California 94720, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural