One Tube is all it Takes

Novagen's STP3 performance with various templates Promega's TNT® system and Novagen's Single Tube Protein™ System 3 combine in vitro transcription and translation into a single-tube reaction. Traditionally, in vitro transcription and translation are carried out as two separate reactions where the RNA reactions is first synthesized using an in vitro transcription system and then added to a translation reaction, of which several are in use today. Carrying out two separate procedures can be time consuming, taking as much as 1--2 days, and entails some risk, since it requires handling labile RNA species in a purification step. However, in the last few years, procedures have been developed that combine the transcription and translation steps into simpler and faster single-tube reactions. This development has opened up a number of applications for this technology, such as studying protein:protein or protein: DNA interactions, looking at protein truncation as an indicator of mutations, and performing functional genomic studies on entire cDNA libraries. Currently, two systems on the market offer one-tube transcription and translation: the TNT® system from Promega and the Single Tube Protein™ System 3 (STP3) from Novagen. Although similar, the two systems have distinct differences. Promega's TNT system is coupled such that the translation and transcription reactions occur at the same time. According to Karen Martin, molecular biology product manager for this system, Promega holds the patent on one-tube, coupled transcription and translation. Promega expanded its product offering from the original one-tube system, introduced at the end of 1992, to include the simplified Quick Coupled version in 1996. The Quick version includes a one-tube master mix that contains all the necessary reagents for the reactions. Ryan noted that while many customers prefer the Quick format, some continue to use the original system because of the flexibility it affords with respect to the type and concentration of label used. Promega offers a range of transcription/translation systems for researchers, including both rabbit reticulocyte lysate and wheat germ extract systems for T7, SP6, or T3 phage polymerase promoters. Promega's systems are designed for transcription/translation from a variety of templates including plasmid, PCR products and cDNA libraries. The recently introduced Novagen STP3 system is a single tube, two-step system; following a transcription reaction, the translation reaction is initiated by the addition of translation mix and label. Corrine Fetherston, product manager for the system, noted that this system is consistent with eukaryotic transcription/translation. She adds that "since RNA is completely transcribed before translation occurs, it is less likely to make truncated proteins." Fetherston also mentioned that in Novagen's studies the STP3 system "worked exceptionally well with PCR products." Dr. Rosemary Jagus, professor at the Center for Marine Biotechnology in Baltimore, Md., has been using in vitro transcription and translation systems for a number of years to study the activity of translation initiation factors. As part of some collaborations, she has used one-tube, in vitro transcription/translation systems to express microgram quantities of protein, such as terrapin aromatase and an archeobacterial protein for characterization. Jagus commented that in transcription/translation systems, circular DNA generally gives the best results, but when linear DNA, such as PCR products, must be used, the T7 promoter works better than the SP6 promoter. Also, she emphasized that for optimal translation, it is important to give adequate thought to the plasmid construction. Since mRNAs are not capped in the reaction, the transcripts from the plasmid DNA should provide an internal ribosome entry site upstream of the translation start site, as provided by the Novagen's pCITE vector. In addition, the cDNA should be cloned in such a way as to minimize potentially inhibitory sequences found in some 5' untranslated regions. These systems are not just a boon to researchers. Pharmaceutical companies are utilizing single-tube systems for high throughput screening of random, recombinant, or select coding regions. In addition, diagnostic and clinical tests, such as the protein truncation test, are being developed around this new platform. For more information concerning the TNT® Quick Coupled Transcription/Translation System, contact Promega at 800-356-9526 or at www.promega.com. For more information concerning the Single Tube ProteinTM System3 (STP3), contact Novagen at 800-526-7319 or www.novagen.com. The author, Paul Diehl can be reached at paul_vfocus@msn.com.

One Tube is all it Takes

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