Title |
An efficient ligation method in the making of an in vitro virus for in vitro protein evolution
|
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Published in |
Biological Procedures Online, October 2002
|
DOI | 10.1251/bpo33 |
Pubmed ID | |
Authors |
Ichiro Tabuchi, Sayaka Soramoto, Miho Suzuki, Koichi Nishigaki, Naoto Nemoto, Yuzuru Husimi |
Abstract |
The "in vitro virus" is a molecular construct to perform evolutionary protein engineering. The "virion (=viral particle)" (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this "viral genome" was demonstrated. |
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Unknown | 21 | 100% |
Demographic breakdown
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Student > Master | 3 | 14% |
Student > Postgraduate | 2 | 10% |
Student > Bachelor | 1 | 5% |
Other | 1 | 5% |
Other | 2 | 10% |
Unknown | 5 | 24% |
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Pharmacology, Toxicology and Pharmaceutical Science | 1 | 5% |
Other | 0 | 0% |
Unknown | 5 | 24% |