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 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.
Shevchuk
Nikolai A
NA
Center for Cancer and Immunology Research, Children's Research Institute, Washington, DC 20010, USA. niash@gwu.edu
Bryksin
Anton V
AV
Nusinovich
Yevgeniya A
YA
Cabello
Felipe C
FC
Sutherland
Margaret
M
Ladisch
Stephan
S
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012799:Sialyltransferases / Q000235:genetics
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D012799:Sialyltransferases; C021343:haematoside synthetase