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1.
PLoS One ; 11(4): e0153158, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27073895

RESUMO

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.


Assuntos
Clonagem Molecular/métodos , DNA Recombinante/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Plasmídeos/metabolismo , DNA Recombinante/economia , DNA Recombinante/genética , DNA Polimerase Dirigida por DNA/economia , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/economia , Plasmídeos/genética , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos
3.
Hist Sci (Tokyo) ; 19(1): 1-18, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19860031

RESUMO

Recombinant DNA technology was developed in the United States in the early 1970s. Leading scientists held an international Asilomar Conference in 1975 to examine the self regulation of recombinant DNA technology, followed by the U.S. National Institutes of Health drafting the Recombinant DNA Research Guidelines in 1976. The result of this conference significantly affected many nations, including Japan. However, there have been few historical studies on the self-regulation of recombinant technologies conducted by scientists and government officials in Japan. The purpose of this paper is to analyze how the Science Council of Japan, the Ministry of Education, Science adn Culture, and the Science and Technology Agency developed self-regulation policies for recombinant DNA technology in Japan in the 1970s. Groups of molecular biologist and geneticists played a key role in establishing guidelines in cooperation with government officials. Our findings suggest that self-regulation policies on recombinant DNA technology have influenced safety management for the life sciences and establishment of institutions for review in Japan.


Assuntos
Congressos como Assunto , DNA Recombinante , Órgãos Governamentais , Guias como Assunto , Pesquisadores , Tecnologia , Congressos como Assunto/história , DNA Recombinante/economia , DNA Recombinante/história , Genética/educação , Genética/história , Órgãos Governamentais/economia , Órgãos Governamentais/história , Órgãos Governamentais/legislação & jurisprudência , Programas Governamentais/economia , Programas Governamentais/educação , Programas Governamentais/história , Programas Governamentais/legislação & jurisprudência , História do Século XX , Japão/etnologia , Pessoal de Laboratório Médico/educação , Pessoal de Laboratório Médico/história , Pessoal de Laboratório Médico/psicologia , Biologia Molecular/educação , Biologia Molecular/história , Pesquisadores/educação , Pesquisadores/história , Pesquisadores/psicologia , Tecnologia/educação , Tecnologia/história
5.
Isis ; 92(3): 541-75, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11810894

RESUMO

In 1973-1974 Stanley N. Cohen of Stanford and Herbert W. Boyer of the University of California, San Francisco, developed a laboratory process for joining and replicating DNA from different species. In 1974 Stanford and UC applied for a patent on the recombinant DNA process; the U.S. Patent Office granted it in 1980. This essay describes how the patenting procedure was shaped by the concurrent recombinant DNA controversy, tension over the commercialization of academic biology, governmental deliberations over the regulation of genetic engineering research, and national expectations for high technology as a boost to the American economy. The essay concludes with a discussion of the patent as a turning point in the commercialization of molecular biology and a harbinger of the social and ethical issues associated with biotechnology today.


Assuntos
DNA Recombinante/história , Biologia Molecular/história , Patentes como Assunto/história , Biotecnologia/economia , Biotecnologia/história , Biotecnologia/legislação & jurisprudência , Comércio/história , DNA Recombinante/economia , História do Século XX , Biologia Molecular/economia , Biologia Molecular/legislação & jurisprudência , Estados Unidos
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