Optimization Of Direct DNA Cloning Technology

The genomes continues to reveal a rich source of biosynthetic pathways.In genetically tractable microorganisms,elucidation and modification of natural product biosynthetic pathways can be achieved by gene knockout or insertion of promoters.However,for genetically intractable microorganisms,to study its natural product biosynthetic pathways,the relevants genes or gene clusters must be cloned and expressed in heterologous hosts.Most of gene clusters of natural products,particularly secondary metabolites,are larger than 10 kb and they can not be cloned by PCR amplification.Conventional genomic library is known to be labor intensive and time consuming and can not satisfy the need for investigation of the potential wealth.In 2012,Jun Fu et al.published in Natue Biotechnology the RecET direct DNA cloning technology.This direct DNA cloning approach is based on the highly efficient linear-linear homologous recombination mediated by Rac prophage protein RecE and its partner RecT.In RecET direct cloning,the homologous recombination can only happen when the cloning vector and the target DNA segment enter into one cell simultanenously.Standard methods for extracting genomic DNA from cells usually produce fragments.The cloning efficiency decreases when the target genomic segment is longer.Because the longer the target DNA segment is,the smaller the proportion of the target segment in the digested genomic DNA is,and the chance that the target segment enters into one cell simultaneously with the linear vector will be lower.When the target genomic segment is longer than 50 kb,the efficiency of RecET cloning is very low and it is very difficult to clone.Therefore,increasing the chance that the target genomic segment and the cloning vector enter into one E.coli cell upon electroporation is critical for improvement of the efficiency of RecET direct cloning.In 2007,Mamie Z Li and Stephen J Elledge published in Nature Methods the SLIC(sequence and ligation-independent cloning)assembly method which allows the in vitro assembly of multiple DNA fragments via annealing of single-stranded DNA overhangs at both ends of each DNA fragment generated by the 3'-5'exonuclease activity of T4 DNA polymerase.Inspired by SLIC assembly,in this study we used exonulcease to treat the mixture of fragmented genomic DNA and the linear vector in vitro to generate single-stranded DNA overhangs at both ends of linear DNA molecules,and join the target genomic fragment and the cloning vector by single-stranded DNA annealing to increase the chance that the target genomic fragment and the cloning vector enter into one cell upon electroporation to improve the efficiency of direct DNA cloning.At first,we compared a series of exonuclases and found that T4 DNA polymerase and some other exonuclases can significantly improve the efficiency of direct DNA cloning,but T4 DNA polymerase is consistently the best.Then,we settled on T4 DNA polymerase and optimized its concentration,the incubation temperature and duration in the in vitro reaction.We also tested effects of the length of homology arms and the copy number of recE/recT on direct cloning efficiency.The conclusions we obtained are(1)the longest homology arm length tested(80 bp)was the best;(2)the direct cloning efficiency was highest when recE/recT from both the plasmid and chromosome(?6 copies)were employed;(3)the optimal in vitro reaction procedure for T4 DNA polymerase is:0.02 U?l-1,25? 1h,75? 20min,50? 30min.In this study,based on the remarkable capacity of full length RecET homologous recombination,we developed the ExoCET(Exonuclease Combined with RecET recombination)direct cloning strategy.The direct cloning efficiency of ExoCET is-200 times higher than RecET and target genomic DNA fragment longer than 100 kb can be directly cloned into a BAC vector at a reasonable efficiency.ExoCET will greatly facilitate the functional genomic research.