ArticleNucleic acids research2009
Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis.
Article in Nucleic acids research, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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5 citing papers in PubMed, 15 citations in OpenAlex.
- Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.Nucleic acids research · 2016Article
- A microfluidic oligonucleotide synthesizer.Nucleic acids research · 2010Article
- Synthetic biology: tools to design, build, and optimize cellular processes.Journal of biomedicine & biotechnology · 2010Review
- TmPrime: fast, flexible oligonucleotide design software for gene synthesis.Nucleic acids research · 2009Article
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Authors and funding
4 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10 degrees C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.
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