In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation

Abstract

A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein of a jellyfish Aequorea victoria was extensively diluted to one molecule per well, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequences obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and Escherichia coli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.

Single molecule transcription profiling with AFM

“…Genes transcribed at low levels, such as regulatory proteins, exert large biological effects from small changes in expression level [13,18,22]. Considerable work with enzymatic amplification, including PCR-based [23][24][25][26][27][28][29][30][31][32][33], multiple displacement amplification-based (MDA) [24,, and RNA polymerase-based [23,[56][57][58][59][60][61][62][63] protocols, has enabled the use of hybridization microarrays and sequence tag methods to characterize low abundance mRNA samples [64][65][66][67][68][69][70][71][72]. This includes several recent reports of message profiling of single cells [73][74][75][76][77][78][79][80][81][82].…”

Increasing PCR Fragment Stability and Protein Yields in a Cell-Free System with Genetically Modified <i>Escherichia coli </i>Extracts

“…Furthermore, linear DNA disappears after a few minutes of incubation. Other groups that have expressed proteins from PCR products have required large amounts of DNA (10-20 g/ml of reaction) but produced only nominal amounts of protein [Burks et al, 1997;Lesley et al, 1991;Merk et al, 2004;Nemetz, 2004;Ohuchi et al, 1998]. …”

A biological treasure metagenome: pave a way for big science

“…Furthermore, in case of gram positive or minority strains, it is known hard to extract genomes through a general process of cell lysis [34,35]. As a solution, stable isotope labeling [36,37] or single molecule PCR [38] has been introduced. Gel fi ltration, agarose plugs and/or electro-elution are also being incorporated into the extraction process of metagenome but there are still many diffi cult problems to be solved [39].…”