Cloning Without Bacteria?

Invitrogen Corp. of Carlsbad, Calif., offers TOPO® Tools for those interested in an alternative to conventional, time-consuming cloning techniques. TOPO Tools provide a relatively fast way of joining various sequence elements, such as promoters and tags, to either or both ends of a PCR-amplified product, without traditional cloning procedures such as ligations, vector manipulations, or Escherichia coli transformation. Rather, this method harnesses the dual catalytic activities of Vaccinia topoisomerase I: Sequence-specific cleavage and nucleic acid joining. Courtesy of InvitrogenInvitrogen'S TOPO Tools procedure. The researcher first PCR-amplifies the gene or gene segment of interest using a proofreading polymerase and primers that contain a specific 11-nucleotide sequence. The resultant product contains the gene of interest flanked on each end by a conserved, five-nucleotide topoisomerase-binding/cleavage site [(C/T)CCTT]. The six additional bases establish directionality for the subsequent topoisomerase-catalyzed joining step, in which sequence elements are specifically added to the 3' and/or 5' ends of the fragment. Like a molecular crane, topoisomerase, covalently attached to the soon-to-be added sequence, conveys its cargo to the specified end of the PCR product. Topoisomerase first creates a single-strand nick in the PCR product, leaving an overhang that is compatible with its bound sequence element. The enzyme then uses its nucleic acid joining activity to fuse the element to the PCR product by joining the compatible ends. The resulting linear construct is then amplified via a secondary PCR reaction using element-specific primers. Invitrogen offers a number of TOPO Tools kits and reagents for in vitro transcription and translation, transient protein expression in mammalian cells, and mammalian two-hybrid assays. "Researchers can now go directly from PCR of the gene of interest to expression analysis in the same day. This technology is useful for research conducted on all scales, and is extremely powerful for high-throughput applications," comments Debora Galasso, Invitrogen's marketing manager of cloning technologies. "Our variation on the mammalian two-hybrid system is especially exciting. When employing more traditional procedures, researchers typically take several days to a week just to construct the clones needed for the analysis; by using the TOPO Tools system, they can obtain results within 24-48 hours." TOPO Tools reagents for in vitro transcription and translation include those for appending a T3 or T7 promoter; a polyadenine sequence at either end of the PCR fragment for sense or antisense transcription; or for inserting a T7 promoter with Kozak sequence at the a 5' end for efficient translation. Mammalian expression elements include a 5' CMV promoter, with or without control elements for regulated expression; a 3' SV40 polyadenylation signal; a 3' BGH polyadenylation signal along with the coding sequence for the V5 epitope, which enables detection of the fusion protein; and a 3' BGH/V5 construct that encodes a histidine tag for detection and purification (See related story on page 37). Reagents for the mammalian two-hybrid system are bundled in a kit. Those wanting a "hard copy" of a given TOPO Tools-generated construct for additional analyses can use a one-step cloning procedure to insert it into a separately sold TOPO-adapted plasmid. "Researchers can use vector-based cloning for only those constructs that they want to use in further studies, and this can be done in parallel while they are expression profiling," notes Galasso. According to Invitrogen, it is unlikely that results obtained using TOPO Tools methodology for any application would be confounded by PCR errors. Galasso explains that, because transfection and expression is from a pool of PCR species rather than from a single clone, the output will reflect the dominant PCR product. Invitrogen is also willing to discuss the possibility of custom services with those interested in TOPO Tools not currently available "off-the-shelf." —Deborah A. Fitzgerald For More Information: Invitrogen Corp. +1 (800) 955-6288 www.invitrogen.com

Cloning Without Bacteria?

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