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Characterization of TNT® T7 Quick for PCR DNA: IntroductionBy Natalie Betz, Ph.D.
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An article in four parts with references characterizing the use of the TNT® T7 Quick for PCR DNA (Cat.# L5540). 1. Introduction |
The importance of matching a genetic sequence with a particular protein function is becoming increasingly important as the amount of genome sequence information increases and becomes readily available to researchers. Multiple approaches are usually required to determine the function of any particular gene. Many of these studies can be performed using protein biochemistry and in vitro expression methodologies, such as in vitro translation.
Using DNA in a coupled transcription/translation format allowed the development of many in vitro expression applications, including gene construct verification, determination of protein function, detection of molecular interactions, detection of post-translational modifications, detection of disease-causing mutations by protein truncation test (PTT) or in vitro synthesized protein assay (IVSP), in vitro expression cloning(a) (IVEC) and ribosome display systems for cell-free protein evolution (1).
PCR(b)-generated DNA has increasingly become the template of choice for TNT® coupled transcription/translation reactions due to the ease of generating and using PCR products directly versus cloning specific targets by conventional means into plasmid vectors that contain genetic expression elements. TNT® T7 Quick for PCR DNA* was optimized for the expression of linear, unpurified PCR products. In comparison, the standard TNT® T7 Quick System* is optimized for plasmid DNA expression.
A T7 phage RNA polymerase promoter is required for transcription initiation from the PCR product DNA template. The T7 promoter may either be amplified from the plasmid vector containing the gene of interest, or the T7 promoter can be designed into the PCR product by addition to the forward or 5´ amplification primer. To ensure efficient translation initiation, a Kozak consensus sequence should also be present. The reverse or 3´ primer typically matches the carboxy terminus of the gene of interest and includes a stop codon (TAA, TGA or TAG). Promega Notes, Issue 74, discusses effective primer design (166 kilobyte pdf file).
Earlier work using TNT® T7 Quick for PCR DNA demonstrated that the system does not require any post-amplification purification of the template DNA and can produce up to five times more protein than other commercially available kits (Technical Manual #TM235). This article further characterizes the use of TNT® T7 Quick for PCR DNA with respect to template attributes, template purity and compatibility with other procedures.
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Figure 1. Agarose gel of PCR products amplified from an 8.7kb cDNA template. |
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Figure 2. In vitro translation of APC PCR products using TNT® T7 Quick for PCR DNA. |
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Figure 3. Forward and reverse primers used for amplification of the APC PCR products. |
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Figure 4. Effect of the ATG start codon context and presence of poly(A) tail on expression of APC PCR products with TNT® T7 Quick for PCR DNA. |
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Figure 5. Expression of 20kDa (Panel A) and 40kDa (Panel B) APC PCR products generated with or without dUTP in PCR. |
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Figure 6. In vitro transcription using the RiboMAX™ System of APC PCR products generated with or without dUTP in PCR. |
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