With the established BioBrick Assembly standards, ligation of different parts has to be accomplished step by step. It can be time-consuming when dealing with multiple fragment ligation. BBF RFC 62 is developed aimed at completing the ligation of multiple fragments quickly and efficiently based on homologous recombination

Chen, Guoqiang

Li, Teng

Shi, Zhenyu

Wang, Ruiyan

DSpace@MIT

BBF RFC 61 Fast multiple gene fragment ligation method based on homologous recombination BBF RFC 62: Fast multiple gene fragment ligation method based on homologous recombination  Requested by Ruiyan WANG, Zhenyu SHI, Teng LI, and Guoqiang CHEN  25 October 2010 1. Purpose With the established BioBrick Assembly standards, ligation of different parts has to be accomplished step by step. It can be time-consuming when dealing with multiple fragment ligation. BBF RFC 62 is developed aimed at completing the ligation of multiple fragments quickly and efficiently based on homologous recombination. 2. Relation to other BBF RFCs This BBF RFC updates BBF RFC 10. 3. Copyright Notice Copyright (C) The BioBricks Foundation (2010). All Rights Reserved. 4. Description This method depends on sequence and ligation-independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro.   Homologous recombination in vivo depends upon a double-stranded break, generation of ssDNA by exonucleases, homology searching by recombinases, annealing of homologous stretches, and repair of overhangs and gaps by enzymes that include resolvases, nucleases and polymerases.  It is possible to generate recombination intermediates in vitro and introduce these into cells to allow the cell endogenous repair machinery to finish the repair to generate recombinant DNA.  The experiment generally includes the following steps: a) Introduce restriction sites and homologous recombination sites to the vector.   BBF RFC 61 Fast multiple gene fragment ligation method based on homologous recombination “H” stands for “Homologous Recombination Site”  b) Generate the vector by cleavage with a restriction enzyme  c) Generate the insert by PCR. d) Treat both the vector and the insert with T4 DNA polymerase in the absence of dNTPs to chew back 5’ strand to reveal ssDNA overhangs. e) Incubate vector and insert with RecA protein and ATP to promoter recombination. f) Transform the products into E.coli.    E X S  P H H BBF RFC 61 Fast multiple gene fragment ligation method based on homologous recombination When dealing with multiple gene fragments, these fragments can be introduce with different homologous recombination sites, then mixed with the vector. Different fragments can be generated as this:  Fragment A : (Homology)1-(n1n1n1…n1n1n1)- (Homology)2 Fragment B: (Homology)2-( n2n2n2…n2n2n2)- (Homology)2 Fragment C: (Homology)2-( n3n3n3…n3n3n3)- (Homology)2 …  5. Author’s Contact Information Ruiyan WANG  ry-wang07@mails.tsinghua.edu.cn  Zhenyu SHI errisy@gmail.com  Teng LI liteng07@mails.tsinghua.edu.cn  Guoqiang CHEN chengq@mail.tsinghua.edu.cn   6.References [1] Mamie Z Li & Stephen J Elledge Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC, NATURE METHODS  VOL.4 NO.3 [2] Sambrook J, Maniatis T, Fritsch E F. Molecular Cloning: a Laboratory Manual. cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd ed., 2001.  

Fast multiple gene fragment ligation method based on homologous recombination

http://dspace.mit.edu/bitstream/1721.1/59803/1/BBFRFC62.pdf

Fast multiple gene fragment ligation method based on homologous recombination

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