Error removal in microchip-synthesized DNA using immobilized MutS.
The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Wen
W
Wan
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Lulu
L
Li
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Qianqian
Q
Xu
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Zhefan
Z
Wang
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Yuan
Y
Yao
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Rongliang
R
Wang
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Jia
J
Zhang
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
Haiyan
H
Liu
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China Department of Biology and Biochemistry, University of Houston, Houston, TX, USA xgao@uh.edu.
Xiaolian
X
Gao
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China hjiong@ustc.edu.cn.
Jiong
J
Hong
D004247:DNA / Q000096:biosynthesis*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015870:Gene Expression; D005813:Genes, Synthetic; D049452:Green Fluorescent Proteins / Q000235:genetics; D055767:Immobilized Proteins / Q000378:metabolism; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010105:Oxygenases / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods
D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; D010105:Oxygenases; C021555:methane monooxygenase; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli