Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences.
Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.
Department of Chemistry, University of Houston, Houston, TX 77004-5003, USA.
Xiaochuan
X
Zhou
Shiying
S
Cai
Ailing
A
Hong
Qimin
Q
You
Peilin
P
Yu
Nijing
N
Sheng
Onnop
O
Srivannavit
Seema
S
Muranjan
Jean Marie
JM
Rouillard
Yongmei
Y
Xia
Xiaolin
X
Zhang
Qin
Q
Xiang
Renuka
R
Ganesh
Qi
Q
Zhu
Anna
A
Matejko
Erdogan
E
Gulari
Xiaolian
X
Gao
D001483:Base Sequence; D005813:Genes, Synthetic; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis* / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction
D009838:Oligodeoxyribonucleotides