Cell-free protein synthesis using multiply-primed rolling circle amplification products.
Utilizing in vitro transcription and translation (IVTT) to produce small quantities of proteins is convenient but requires a significant supply of pure template DNA. This can be cumbersome, particularly when the method is used for many different templates in a high-throughput manner. Multiply-primed rolling circle amplification (RCA) with [#x03C6]29 DNA polymerase is a simple way to generate large amounts of DNA; however, the products of this amplification method have interruptions in both strands and branched structures. In this study, we tested whether RCA-generated DNA can serve as the template for in vitro transcription. We found that RCA DNA[#x02013]generated transcripts work in coupled in vitro translation with nearly the same efficiency (per nanogram of DNA) as those obtained from purified plasmid. We propose a convenient, single-tube format for template amplification, transcription, and translation.
Kumar
Gyanendra
G
GE Healthcare Bio-Sciences Corp. Advanced Systems Division, Piscataway, NJ, USA. kumargyan@hotmail.com
Chernaya
Galina
G
D017103:Bacillus Phages / Q000201:enzymology; D002474:Cell-Free System; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D013696:Temperature; D013698:Templates, Genetic; D013997:Time Factors
D004270:DNA, Circular; D004279:DNA, Viral; D049452:Green Fluorescent Proteins; D006639:Histidine; D004259:DNA-Directed DNA Polymerase