aspects of the vector. The vector has been engineered to be a
linearized plasmid with 3' deoxythymidine (T) overhangs that is activated
by being covalently bonded to topoisomerase I. The 3' A overhangs
of the PCR product complement the 3' T overhangs of the vector and allow
for fast ligation with the already present topoisomerase I. The plasmid
can then be transformed into competent bacterial cells.2
Other useful features of the vector are its ampicillin and kanamycin resistance markers, lacZ reporter gene, T7 promoter, EcoR I sites flanking the PCR insertion site, and the f1 origin of replication. The ampicillin and kanamycin resistance inserts in the plasmid allow for quick selection of bacterial colonies that take up the vector plasmid during transformation. The lacZ gene in bacteria cause colonies to have a blue color. If PCR DNA is inserted in the vector it will insert in the middle of the lacZ gene causing the colonies to be white and easily selected. The T7 promoter region in the vector allows for in vitro RNA transcription/translation by the T7 phage. If T7 phage infects the bacteria it will make proteins from the DNA sequence of the vector and insert which allows scientists to sequence the subsequent protein and therfore DNA sequence of the insert. The EcoR I sites on either side of the insert enable the insert to be easily removed by EcoR I restriction enzymes. The f1 origin of replication is a necessary component of the plasmid and makes single-strand rescue possible.2
The entire TOPO Cloning process is shown below (Fig. 2) and can be done in only five
minutes at room temperature with 95% efficiency. This is much
faster and less complicated than the PRIME PCR Cloner Cloning System we
used in lab and most other cloning procedures. TOPO Cloning system
is ideal for scientists that need to quickly clone PCR products into bacteria.
1 Campbell NA, Biology. 4th ed. Menlo Park, CA: The Benjamin/Cummings Publishing Company, Inc.; 1996. p 379-380.
2 Hoover C, 1998 July 8. Invitrogen
web catalog. <http://www.invitrogen.com/pdf_manuals/topota_man.pdf
> Accessed 1999 Feb 14.
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