Published online 22 January 2004
Nucleic Acids Research, 2004, Vol. 32, No. 2 e19
© 2004 Oxford University Press
Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously
1 Center for Cancer and Immunology Research, Children’s Research Institute, Washington, DC 20010, USA, 2 Institute for Biomedical Sciences/Program in Molecular and Cellular Oncology, 3 Department of Pharmacology, The George Washington University, Washington, DC 20037, USA, 4 Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA and 5 Institute of Bioorganic Chemistry, Novosibirsk 630090, Russia
*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{at}gwu.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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.
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