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1.
Nucleic Acids Res ; 46(2): 538-545, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29237070

RESUMO

Biophysical properties of DNA such as its longitudinal and torsional persistence length govern many processes and phenomena in biology, DNA nanotechnology and biotechnology. It has, for example, long been known that the circularization efficiency of short DNA fragments shows a periodic pattern where fragments with integer helical turns circularize much more efficiently than those with odd helical half turns due to stronger stacking of duplex ends. Small DNA circles can serve as templates for rolling circle amplification (RCA), which is a common and extremely robust amplification mechanism for nucleic acids. We discovered a strong template length-dependent amplification efficiency bias of RCA with the same periodicity as B-DNA. However, stacking cannot explain the mechanism behind this bias as the presence of the polymerase in the bifurcation fork inhibits base stacking of ends. Instead, coarse-grained molecular dynamics simulations imply that different amplification efficiencies come from a varying fraying probability of the last two downstream base pairs. We conclude that an increased strain-promoted fraying probability can increase the polymerization rate compared to a relaxed template.


Assuntos
DNA Circular/genética , Amplificação de Genes , Técnicas de Amplificação de Ácido Nucleico/métodos , Moldes Genéticos , DNA Polimerase III/química , DNA Polimerase III/metabolismo , Replicação do DNA/genética , DNA Circular/química , DNA Circular/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos
2.
Nat Commun ; 6: 8634, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26567534

RESUMO

Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.


Assuntos
DNA de Cadeia Simples , Biblioteca Gênica , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligonucleotídeos , Cromatografia Líquida de Alta Pressão , Hibridização in Situ Fluorescente , Nanoestruturas , Nanotecnologia , Técnicas de Amplificação de Ácido Nucleico/economia , Análise de Sequência com Séries de Oligonucleotídeos/economia , Biologia Sintética
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