TopDown real-time gene synthesis.
This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer primers is designed to be ∼8°C lower than that of the assembly oligonucleotides, and (2) different annealing temperatures are utilized to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. This method eliminates the interference between polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. Additionally, the TopDown gene synthesis has been combined with the LCGreen I DNA fluorescence dye in a real-time gene synthesis approach for investigating the stepwise efficiency and kinetics of PCR-based gene synthesis. The obtained real-time fluorescence signals are compared with gel electrophoresis results to optimize gene synthesis conditions.
Institute of Bioengineering and Nanotechnology, The Nanos, Singapore.
Mo Chao
MC
Huang
Wai Chye
WC
Cheong
Hongye
H
Ye
Mo-Huang
MH
Li
D003199:Computer Systems; D017076:Computer-Aided Design; D003362:Cost-Benefit Analysis; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005813:Genes, Synthetic / Q000235:genetics*; D007202:Indicators and Reagents / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D044366:Transition Temperature
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D004247:DNA