Format

Send to

Choose Destination
Anal Biochem. 2017 Aug 1;530:5-8. doi: 10.1016/j.ab.2017.04.018. Epub 2017 Apr 28.

Sequence-independent cloning methods for long DNA fragments applied to synthetic biology.

Author information

1
Programa de Pós Graduação em Saúde e Meio Ambiente, Universidade da Região de Joinville, UNIVILLE, Joinville, SC 89201-972, Brazil. Electronic address: nenidalo@yahoo.com.br.
2
Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, INCT do Bioetanol, Av. do Café S/N, Ribeirão Preto, SP 14040-903, Brazil.
3
Programa de Pós Graduação em Saúde e Meio Ambiente, Universidade da Região de Joinville, UNIVILLE, Joinville, SC 89201-972, Brazil.

Abstract

Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.

KEYWORDS:

Circular polymerase extension cloning (CPEC); Isothermal NEBuilder; Multiple DNA fragments; PCR fusion-based; Synthetic biology

PMID:
28461174
DOI:
10.1016/j.ab.2017.04.018
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center