DESIGN AND CONSTRUCTION OF TRANSCRIPTION TEMPLATE FOR SYNTHESIZING CELL-FREE PROTEIN, AND DILUTION BATCH-TYPE METHOD OF SYNTHESIZING CELL-FREE WHEAT GERM PROTEIN BY USING THE SAME

摘要

Primers, primer sets, and methods using same to design and construct a transcription template for cell-free protein synthesis that can be widely used, has a high translation template activity, and can be simply constructed, including a 3'-side primer for PCR and a 5'-side primer for PCR are provided. The 3'-side primer for PCR is a polynucleotide comprising a base sequence characteristic to each vector capable of forming a complementary strand with a base sequence present between a transcription terminator sequence of a marker gene, such as a drug resistance gene, of a vector into which a gene was inserted and Ori. The 5'-side primer for PCR comprises polynucleotide (A) that has a sequence complementary to a base sequence containing at least a part of a promoter functional site from the 5'-end of a promoter and has a base sequence that does not contain a base sequence complementary to at least a part of a RNA polymerase-recognizing site of the 3'-end of the promoter, and polynucleotide (B) that has a base sequence containing a base sequence complementary to at least a part of the RNA polymerase-recognizing site of the 3'-end of the promoter and has a sequence complementary to a base sequence that does not contain at least a part of a promoter functional site from the 5'-end of the promoter, and the base sequences of the two primers for PCR satisfy the condition that any transcription does not occur from a DNA constructed with one of the primers. Also, a method for constructing a transcription template using these primers, a transcription template obtained by the construction method, and a protein synthesis method using an mRNA synthesized using the transcription template as a translation template, are provided.