My cry of frustration echoed down the hall. My latest attempt to subclone five important PCR products had failed yet again. After two months of fruitless effort, I was at my wit's end until a colleague suggested I try the TOPO TA Cloning® Kit from Invitrogen.
"Look," he said, "all you need to do is to combine 1 microliter of your PCR reaction and 1 microliter of the pCR2.1-TOPO® vector, add 3 microliters of water, and wait 5 minutes. Then transform your competent cells. In the morning, you will have more white colonies than you will know what to do with." As proof, he showed me a petri dish with six blue colonies surrounded by hundreds of white ones.
I was sold on the spot. I promptly borrowed his TOPO TA Cloning kit and set about subcloning those pesky PCR products.
pCR2.1 Topo Map
At the heart of the TOPO TA Cloning kit beats vaccinia DNA topoisomerase, a 314-amino acid, virus-encoded eukaryotic type I topoisomerase (S. Shuman, S., J. Biol. Chem., 269: 32678-864, 1994). Topoisomerase binds to double-stranded DNA and cleaves it at a CCTTT cleavage site like a restriction endonuclease. The enzyme then remains covalently bound to the DNA and functions to religate a DNA fragment with compatible overhangs, creating a recombinant molecule.
Invitrogen has taken advantage of topoisomerase's unique properties by combining it with a specially designed plasmid, creating the topoisomerase-activated pCR2.1-TOPO vector. Supplied in linearized form with single, 3'-T overhangs, the pCR2.1-TOPO vector exploits the 3'-A overhangs attached to the ends of Taq-amplified PCR products for direct, rapid ligations. Ligation reactions using PCR products are complete in 5 minutes at room temperature, producing cloned DNA that is immediately available for transformation. Invitrogen reports cloning efficiencies (the number of white colonies divided by the total number of colonies) between 89% and 99% depending on the PCR product size, with most efficiencies in excess of 95%.
Mary Tulipani-Cassoni, Invitrogen's TOPO TA Cloning kit product manager, is very excited about the TOPO-Cloning technology. "This is a revolutionary new way of cloning," she says. "The TOPO TA Cloning kit has been very well received by our customers. In fact, we are currently applying TOPO-Cloning technology to some of our most popular expression vectors, as well as developing TOPO-Cloning vectors for a variety of applications. For example, we have recently introduced the TOPO XL PCR Cloning® kit for cloning long PCR fragments between 3 and 10 kb in size. This kit clones a 7 kb fragment with 80% efficiency and is the first kit on the market designed for cloning long PCR products." Additionally, Invitrogen has just released the pBAD TOPO TA Cloning® Kit, the Eukaryotic TOPO TA Cloning® Kit, and the pMT/VS-His TOPO-Cloning® Kit for cloning and expression of Taq-amplified PCR products in E. coli, mammalian cells, and insect cells, respectively. Invitrogen has also undertaken the task of cloning and expressing all 6,000 S. cerevisiae open reading frames (YORF's) at the rate of 190 per day using the TOPO-Cloning technology.
The TOPO TA Cloning Kit comes well supplied with the linearized pCR2.1-TOPO vector, One-ShotTM TOP10® competent cells, sequencing primers, and the necessary reagents (except Taq) for a control PCR cloning reaction as well as pUC18 plasmid DNA for use as a transformation control. In addition to its rapid and efficient subcloning capabilities, the pCR2.1-TOPO vector features flanking EcoR I sites for easy insert excision, both kanamycin and ampicillin selection, the lacZ gene for blue/white colony screening, M13 forward and reverse priming sites for sequencing applications, the T7 promoter/primer for in vitro RNA transcription/translation and sequencing, and the f1 origin of replication for single-strand rescue. Kits for 20 or 40 reactions are available, with list prices beginning at $292.00.
For more information, contact Invitrogen at 800-955-6288 or visit the company's web site at www.invitrogen.com. Michael Brush is an Associate Scientist at Hitachi Chemical Company in Irvine, Calif. He can be reached online at MDBrush@compuserve.com.