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Error removal in microchip-synthesized DNA using immobilized MutS.

ABSTRACT: 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.

SUBMITTER: Wan W

PROVIDER: S-EPMC4081059 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Error removal in microchip-synthesized DNA using immobilized MutS.

Wan Wen W Li Lulu L Xu Qianqian Q Wang Zhefan Z Yao Yuan Y Wang Rongliang R Zhang Jia J Liu Haiyan H Gao Xiaolian X Hong Jiong J

Nucleic acids research 20140514 12

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 sp ...[more]

PMID: 24829454

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Project description:PurposePer- and polyfluoroalkyl substances (PFAS) have been found to be widespread, extremely persistent and bioaccumulative with toxicity tendencies. Pre-synthesized nanocomposite-activated carbons, referred to, as physically activated maize tassel silver (PAMTAg) and chemically activated maize tassel silver (CAMTAg) were utilized in the present study. They were used for the removal of 10 PFAS from aqueous solutions.MethodsThe nanocomposite-activated carbons were characterized via scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, Brunauer Emmett Teller (BET) and other techniques. Batch equilibrium experiments were conducted in order to investigate the effects of solution pH, adsorbent dosage, initial PFAS concentration and temperature on the removal of PFAS using PAMTAg and CAMTAg. Langmuir and Freundlich adsorption isotherm models were used to analyse the equilibrium data obtained.ResultsMaximum adsorption capacities of 454.1 mg/g (0.91 mmol/g) and 321.2 mg/g (0.78 mmol/g) were recorded for perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA), respectively using CAMTAg. The values recorded for the Gibbs' free energy (ΔG°) for the adsorption of PFOS and PFOA onto PAMTAg and CAMTAg were negative; PFOS (-9.61, -9.99 and - 10.39), PFOA (-8.77, -9.76 and - 10.21) using PAMTAg; and PFOS (-13.70, -12.70 and - 12.37), PFOA (-12.86, -12.21 and - 11.17) using CAMTAg. Therefore, the adsorption processes were spontaneous and feasible. The values recorded for enthalpy (ΔH°) (kJ/mol) for the adsorption of PFOS (-26.15) and PFOA (-35.86) onto CAMTAg were negative, indicating that the adsorption mechanism is exothermic in nature. Positive values were recorded for ΔH° for the adsorption of PFOS (2.32) and PFOA (12.69) onto PAMTAg, indicative of an endothermic adsorption mechanism. Positive entropy (ΔS°) values (0.04 and 0.07) were recorded for PFOS and PFOA using PAMTAg; whereas negative values (-0.04 and - 0.08) were recorded for ΔS° using CAMTAg. A positive ΔS° indicates an increase in randomness of the adsorbate at the solid-solution interface and the reverse is the case for a negative ΔS°.ConclusionThe interplay of electrostatic attraction and hydrophobic interactions enabled the removal of PFAS using PAMTAg and CAMTAg. Findings suggest that PAMTAg and CAMTAg are effective for the removal of PFAS from aqueous media and are good alternatives to commercially available activated carbons.Graphical abstractSupplementary informationThe online version contains supplementary material available at 10.1007/s40201-020-00597-3.

Dataset Information

Error removal in microchip-synthesized DNA using immobilized MutS.

Publications

Error removal in microchip-synthesized DNA using immobilized MutS.

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