Journal ArticleDOI
Man-made cell-like compartments for molecular evolution
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TLDR
It is demonstrated the linkage of genotype to phenotype in man-made compartments using a model system and a selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion.Abstract:
Cellular compartmentalization is vital for the evolution of all living organisms. Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype. We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions. These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts. A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product. In other compartments, substrates attached to genes that do not encode catalysts remain unmodified. Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product. We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system. A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion. Genes encoding HaeIII methyltransferase were selected from a 10
7
-fold excess of genes encoding another enzyme.read more
Citations
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Journal ArticleDOI
Genome sequencing in microfabricated high-density picolitre reactors
Marcel Margulies,Michael Egholm,William E. Altman,Said Attiya,Joel S. Bader,Lisa A. Bemben,Jan Berka,Michael S. Braverman,Yi-Ju Chen,Zhoutao Chen,Scott Dewell,Lei Du,J. M. Fierro,Xavier V. Gomes,Brian C. Godwin,Wen He,Scott Edward Helgesen,Chun Heen Ho,Gerard P. Irzyk,Szilveszter C. Jando,Maria L. I. Alenquer,Thomas P. Jarvie,Kshama B. Jirage,Jong-Bum Kim,James R. Knight,Janna R. Lanza,John H. Leamon,Steven Lefkowitz,Ming Lei,Jing Li,Kenton Lohman,Hong Lu,Vinod Makhijani,Keith Mcdade,Michael P. McKenna,Eugene W. Myers,Elizabeth Nickerson,John Nobile,Ramona Plant,Bernard P. Puc,Michael T. Ronan,George T. Roth,Gary J. Sarkis,Jan Fredrik Simons,John Simpson,Maithreyan Srinivasan,Karrie R. Tartaro,Alexander Tomasz,Kari A. Vogt,Greg A. Volkmer,Shally H. Wang,Yong Wang,Michael P. Weiner,Pengguang Yu,Richard F. Begley,Jonathan M. Rothberg +55 more
TL;DR: A scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments with 96% coverage at 99.96% accuracy in one run of the machine is described.
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Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome
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Applications of next-generation sequencing technologies in functional genomics.
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Single-cell analysis and sorting using droplet-based microfluidics
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TL;DR: A droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells, and a binding assay for detecting antibodies secreted from single mouse hybridoma cells is detailed.
References
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Bergey's Manual of Systematic Bacteriology
TL;DR: BCL3 and Sheehy cite Bergey's manual of determinative bacteriology of which systematic bacteriology, first edition, is an expansion.
Journal ArticleDOI
Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface
TL;DR: Foreign DNA fragments can be inserted into filamentous phage gene III to create a fusion protein with the foreign sequence in the middle that is incorporated into the virion, which retains infectivity and displays the foreign amino acids in immunologically accessible form.
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Phage antibodies: filamentous phage displaying antibody variable domains
TL;DR: It is shown that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage can be isolated after affinity chromatography.
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DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.
TL;DR: A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported, and mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology.
Journal ArticleDOI
Quantitation of mRNA by the Polymerase Chain Reaction
TL;DR: This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA.
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