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Enzymatic Synthesis of Chimeric DNA Oligonucleotides by in Vitro Transcription with dTTP, dCTP, dATP, and 2'-Fluoro Modified dGTP.
- Source :
-
ACS synthetic biology [ACS Synth Biol] 2021 Jul 16; Vol. 10 (7), pp. 1625-1632. Date of Electronic Publication: 2021 Jun 10. - Publication Year :
- 2021
-
Abstract
- Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an in vitro transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed in vitro evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination. Transcribed chimeric oligonucleotides were tested in PCR, and the quality of amplification products as well as fidelity of oligonucleotide synthesis were assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts contain significantly fewer deletions and insertions compared to chemically synthesized counterparts and can successfully serve as PCR primers, making the evolved enzymes superior for simple and cheap one-pot synthesis of multiple chimeric DNA oligonucleotides in parallel using a plethora of premixed templates.
- Subjects :
- Deoxyguanine Nucleotides chemistry
Substrate Specificity
DNA-Directed RNA Polymerases metabolism
Deoxyadenine Nucleotides genetics
Deoxycytosine Nucleotides genetics
Deoxyguanine Nucleotides genetics
Deoxyribonucleotides genetics
Fluorine chemistry
Synthetic Biology methods
Thymine Nucleotides genetics
Transcription, Genetic
Viral Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 2161-5063
- Volume :
- 10
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- ACS synthetic biology
- Publication Type :
- Academic Journal
- Accession number :
- 34110794
- Full Text :
- https://doi.org/10.1021/acssynbio.1c00112