Bioscience Technology
100 Enterprise Drive
Rockaway, NJ, 07866
|
 |
Simultaneous Insertion Of 2, 3, Or 4 DNA Fragments Into A Single Vector
by Federico Katzen, Brian Dalby, Julia Fletcher, Jon Chesnut, and Wieslaw Kudlicki
MultiSite Gateway Pro (Invitrogen Corp., Carlsbad, CA) facilitates recombination of up to 4 fragments into a single vector. The method is based on the use of 6 novel DONR vectors and a total of 5 different specific Gateway recombination sites. DNA fragments can be simultaneously cloned into a myriad of standard Gateway destination vectors.
Gateway utilizes engineered site-specific recombination sites derived from the
bacteriophage l. Four types of sites are involved in two reactions: the BP reaction,
attB x attP attL + attR and the LR reaction, attL x attR attB + attP(1) (Figure
1A). Positive and negative selection strategies assure that desired recombinants
are recovered at a rate higher than 90% for individual recombination reactions.
In the MultiSite system, the same mechanism used to transfer single DNA elements
from vector to vector is employed to assemble multiple fragments (Figure 1B).
In a more complex permutation, hybrid att sites have been generated that allow
for a wider range of recombination partners, while maintaining the efficiency
and specificity characteristic of the simpler permutations(2) (Figure 1B). This
latter strategy added 5’ and 3’ elements at both ends of Gateway entry clones.
Among other applications, the kit allows mixing and matching of promoters and
ORFs and evaluation of various purification tags.
Due to the equations in this article, we have made a PDF
available for you to download.
|
|
