This page is for informational purposes only.
All questions about the TOPO TA Cloning System should be directed to:

    Invitrogen's TOPO TA Cloning System (TOPO Cloning) is a fast, efficient method for cloning polymerase chain reaction (PCR) products into a plasmid vector.  To understand TOPO Cloning's design features we must first briefly review the mechanism and products of PCR.
    PCR is a method which essentially isolates a specific segment of DNA by amplification.  PCR uses specific primers which allow Taq DNA polymerase to make copies of only a specific segment. Taq DNA polymerase is used for its ability to withstand the high heat (95C) necessary for the many cycles of PCR.  A unique aspect of Taq DNA polymerase is that it adds a single deoxyadenosine (A) to the 3' ends of PCR products.  The resulting products of PCR are many copies of a specific DNA sequence with 3' A overhangs.1
    TOPO Cloning ligates the PCR product into the pCR 2.1-TOPO plasmid vector (vector) with topoisomerase I (Figure 1).  This can be done quickly and efficiently because of the unique

Figure 1. Ligation of PCR product into vector.2

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


Figure 2. The entire TOPO Cloning system.2

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.

References

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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