Construction of long DNA molecules using long PCR‐based fusion of several fragments simultaneously

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and o...

Full description

Saved in:

Bibliographic Details
Published in:	Nucleic acids research Vol. 32; no. 2; p. e19
Main Authors:	Shevchuk, Nikolai A., Bryksin, Anton V., Nusinovich, Yevgeniya A., Cabello, Felipe C., Sutherland, Margaret, Ladisch, Stephan
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 01-01-2004 Oxford Publishing Limited (England)
Subjects:	Base Sequence DNA - chemistry DNA - genetics DNA, Recombinant - chemistry DNA, Recombinant - genetics Green Fluorescent Proteins Humans Luminescent Proteins - genetics Molecular Sequence Data NAR Methods Online Polymerase Chain Reaction - methods Sequence Analysis, DNA Sialyltransferases - genetics
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in‐frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.
Bibliography:	local:gnh014 ark:/67375/HXZ-B2098J2G-N To whom correspondence should be addressed at Center for Cancer and Immunology Research, Children’s Research Institute, 111 Michigan Avenue NW, Washington, DC 20010, USA. Tel: +1 202 884 5802; Fax: +1 202 884 3929; Email: niash@gwu.edu  The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors istex:FDE77FE86E1EF4E43B4AE4F7A61EF2F191B121EE Received November 10, 2003; Revised and Accepted December 2, 2003 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 To whom correspondence should be addressed at Center for Cancer and Immunology Research, Children’s Research Institute, 111 Michigan Avenue NW, Washington, DC 20010, USA. Tel: +1 202 884 5802; Fax: +1 202 884 3929; Email: niash@gwu.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
ISSN:	0305-1048 1362-4962 1362-4962
DOI:	10.1093/nar/gnh014