Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method

Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short...
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Autor*in:	Lendl Tan [verfasserIn] Emily J. Strong [verfasserIn] Kyra Woods [verfasserIn] Nicholas P. West [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2018

Schlagwörter:	Molecular cloning Low cost High efficiency Simple Ligation-independant High fidelity

Übergeordnetes Werk:	In: PeerJ - PeerJ Inc., 2013, 6, p e5146(2018)
Übergeordnetes Werk:	volume:6, p e5146 ; year:2018

Links:	Link aufrufen Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.7717/peerj.5146

Katalog-ID:	DOAJ016913884

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authorswithroles_txt_mv	Lendl Tan @@aut@@ Emily J. Strong @@aut@@ Kyra Woods @@aut@@ Nicholas P. West @@aut@@
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callnumber-first	Q - Science
author	Lendl Tan
spellingShingle	Lendl Tan misc QH301-705.5 misc Molecular cloning misc Low cost misc High efficiency misc Simple misc Ligation-independant misc High fidelity misc Medicine misc R misc Biology (General) Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
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topic_title	QH301-705.5 Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method Molecular cloning Low cost High efficiency Simple Ligation-independant High fidelity
topic	misc QH301-705.5 misc Molecular cloning misc Low cost misc High efficiency misc Simple misc Ligation-independant misc High fidelity misc Medicine misc R misc Biology (General)
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title	Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
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title_full	Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
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title_auth	Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
abstract	Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3′→5′ exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.
abstractGer	Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3′→5′ exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.
abstract_unstemmed	Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3′→5′ exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.
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title_short	Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
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Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method

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