The techniques of modern molecular biology have made possible the manipulation of DNA as well as the other cellular components expressed from the genes contained in the DNA of an organism. In living cells, DNA is transcribed to make mRNA which is then used as a template, in a process called translation, to make a protein whose sequence is determined by the DNA. In developing the modern tools of molecular biology, research has been directed toward ways to perform various steps of the transcription or translation processes in vitro under controlled conditions and with defined inputs. These procedures mimic, in essence, similar processes that occur in a much more heterogeneous mixture in living cells.
Even before the advent of modern recombinant technology, cell extracts were developed which allowed the synthesis of protein in vitro from purified mRNA transcripts. Since that time, several systems have become widely available and are used for the study of protein synthesis and RNA structure and function. To synthesize a protein under investigation, a translation extract must be "programmed" with an mRNA corresponding to the gene or protein under investigation. The mRNA is most often added exogenously in purified form. Historically, such mRNA templates were purified from natural sources or, using more recently developed technologies, prepared synthetically from cloned DNA using bacteriophage RNA polymerases in an in vitro reaction. The preparation of such mRNAs, even by in vitro synthesis, remains a rather tedious process, and this difficulty has limited the practical utility of in vitro translation for a number of applications.
There has consequently been a significant effort in a number of laboratories to develop either coupled or complementary transcription and translation systems which carry out the synthesis of both RNA and protein in the same reaction, beginning with input DNA. These extracts must contain all the components necessary both for transcription (to produce mRNA) and for translation in a single system. In such a system, the input is DNA, which is normally much easier to obtain than RNA and much more readily manipulable. The first such coupled system was based on a bacterial extract. Lederman and Zubay, Biochim. Biophys. Acta, 149:253 (1967). Since prokaryotes normally carry out a coupled reaction within their cytoplasm in any event, this system closely reflected the in vivo process and remains widely used for the study of prokaryotic genes. However, this system is generally not useful for eukaryotic genes, due to its inefficiency and relatively high nuclease content.
Eukaryotic extracts have also been defined that use exogenously added E. coli RNA polymerase or wheat germ RNA polymerase to transcribe exogenous DNA. These systems have had limited success for the general study of eukaryotic genes, due to their low efficiency, and to the fact that they were developed and used prior to the widespread success of cDNA cloning techniques. Other coupled systems have been developed for the study of viral protein synthesis, but are not generally useful for non-viral templates.
In the mid-1980s, the development of highly efficient in vitro transcription systems, particularly ones using phage polymerases such as T7, SP6, and T3, allowed systems to be defined to more efficiently translate cloned mRNA sequences in vitro using translation extracts from wheat germ and rabbit reticulocytes. Perara and Lingappa showed that SP6 RNA polymerase transcription reactions could be added directly to reticulocyte lysate for the production of protein, an insight which illuminated the need to purify the mRNA prior to translation. J. Cell Biol. 101:2292-2301 (1985). Later other workers showed that the transcription and translation could be coupled in reticulocyte lysate by including a phage polymerase and appropriate transcriptional co-factors in the reaction. Spirin et al., Science 242:1162-1164 (1988); Craig et al., Nucleic Acids Res. 20:4987-4995 (1992). More recently, U.S. Pat. No. 5,324,637 describes a coupled transcription and translational system, using reticulocyte lysate and including a phage polymerase, in which the coupling of the two reactions is facilitated by specific conditions, notably the concentration of magnesium ions, which permit both transcription and translation to occur in the same reaction.
Although the coupled approach for transcription and translation systems is useful for many proteins, translation efficiencies can vary widely depending on the type of DNA template which is used (e.g., supercoiled plasmid DNA or linear DNA). In addition, the amount of mRNA synthesized in a coupled reaction is difficult to control under most coupled conditions, such as those described in the aforesaid U.S. Pat. No. 5,324,637. Since the efficiency and fidelity of translation are dependent upon the amount of mRNA added to the reaction, a possible explanation for the undesirable variability of results in a coupled system, in which the reactions occur simultaneously, is that transcription is not consistent between various templates under coupled conditions. Moreover, coupled systems exhibit a marked dependence on the magnesium concentration for the translation efficiency of various templates.