Registry of standard biological parts/PCR-based assembly
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- Automated PCR primer design for worm genes - This is a good example of a simple CAD tool to encourage assembly. You could imagine something similar to auto-generate the primers necessary for PCR-based assembly of standard parts.
- Sauer:Stitching Genes by PCR
- patent on some of this stuff
Contents |
Demo
Going to get a better feel for this by doing a few biobrick constructions using PCR-assembly
- <bbpart>J04500</bbpart>+<bbpart>I13501</bbpart> = <bbpart>J04450</bbpart>
- <bbpart>R0010</bbpart>+<bbpart>I13504</bbpart> = <bbpart>J04430</bbpart>
- <bbpart>F2620</bbpart>+<bbpart>P1010</bbpart>+<bbpart>E0240</bbpart>
- will try this one as a 3-way
UDG
Can use UDG (NEB USER enzyme) to replace all restriction digests and could also eliminate secondary PCRs also. You would just need to design your oligos to contain a U in the right location, assuming that there'll be a T somewhere in the vacinity of the ends of the parts you are trying to combine.
In-Fusion
- Clontech In-Fusion PCR cloning -- this could be used for the last step so that we don't need to do a digest/ligation at any point in the process.
- Seamless cloning and gene fusion review [1]
- Useful forum discussion
- Patents: 1, 2 (might be same as 1, title same though filing date is later), 3
- This reference describes a system similar to In-Fusion using the vaccinia virus DNA polymerase [2]
References
- Lu Q. . pmid:15780712.
- Hamilton MD, Nuara AA, Gammon DB, Buller RM, and Evans DH. . pmid:17158165.
- Horton RM, Cai ZL, Ho SN, and Pease LR. . pmid:2357375.
- Yon J and Fried M. . pmid:2748349.
- Karreman C. . pmid:9591118.
- Wang HL, Postier BL, and Burnap RL. . pmid:12139251.
- Geu-Flores F, Nour-Eldin HH, Nielsen MT, and Halkier BA. . pmid:17389646.
- Donahue WF, Turczyk BM, and Jarrell KA. . pmid:12235397.
- Shevchuk NA, Bryksin AV, Nusinovich YA, Cabello FC, Sutherland M, and Ladisch S. . pmid:14739232.
- McDonald WF and Traktman P. . pmid:7950389.
