Novel method for high-throughput colony PCR screening in nanoliter-reactors.
We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20,000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100,000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.
Walser
Marcel
M
ETH Zurich, Institute of Process Engineering, BioProcess Laboratory (BPL), Zurich, Switzerland.
Pellaux
Rene
R
Meyer
Andreas
A
Bechtold
Matthias
M
Vanderschuren
Herve
H
Reinhardt
Richard
R
Magyar
Joseph
J
Panke
Sven
S
Held
Martin
M
D000464:Alginates / Q000737:chemistry; D001483:Base Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D002365:Manihot / Q000235:genetics; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
D000464:Alginates