Journal: Nucleic Acids Research
Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs
doi: 10.1093/nar/gkag236
Figure Lengend Snippet: NSM enables multiple consecutive and user-constrained DNA mutations. ( A ) Illustration of the NSM workflow when starting from a ExoChase-treated circular DNA molecule. Illustration created with BioRender.com . ( B ) An ExoChase-treated pUC19 plasmid (Fig. ) containing a CcdB toxin gene was incubated with the translesion Sulfolobus DNAP IV (TLS DNAP), with or without the flap endonuclease FEN1, and a nucleotide mixture comprised of dPTP, dRTP, 8-Oxo-dGTP, and 2-Hydroxy-dATP. T4 DNA ligase was added to judge the ability of TLS DNAP alone, or TLS DNAP and FEN1 together, to create a ligatable DNA-nick, i.e. allow for the creation of covalently closed plasmid molecules. The site-specific nickase Nt.BbvCI was then used to introduce a DNA-nick in the opposite wild-type strand following a Proteinase K enzymatic inactivation. ( C ) An ExoChase-treated pUC19 plasmid (Fig. ) containing a CcdB toxin gene was incubated with TLS DNAP and FEN1 together with a nucleotide mixture comprised of dPTP, dRTP, 8-Oxo-dGTP, and 2-Hydroxy-dATP, with or without 1 mM MnCl 2 . T4 DNA ligase was added to judge the ability of manganese to increase the incorporation rate of mutagenic nucleotides by TLS DNAP, which would decrease the efficiency that T4 DNA ligase could ligate the DNA backbone. The pUC19 plasmid was mutated by NSM ( D ) without, or ( E ) with MnCl 2 added. The unmutated wild-type DNA strand was then replaced with the complementary mutated DNA-strand using a reverse-strand ExoChase reaction (revExoChase). NSM-mutated plasmids were transformed into CcdB-sensitive E. coli cells to select for plasmids with a mutation in the ccdB toxin gene. ( F ) The number of consecutive mutational changes that can be created by NSM during a 10 min NSM fill-in reaction using TLS DNAP, FEN1 and 5 µM dPTP, 10 µM 2-Hydroxy-dATP and 100 µM of both dRTP and 8-Oxo-dGTP, with ( n = 47), or without ( n = 47) the addition of 1 mM MnCl 2 . The ccdB gene of surviving E. coli cells transformed with NSM-mutated plasmid was sequenced by single-colony Sanger sequencing. The mutated region was defined as the distance (nt) between the first and last observed mutation in the ccdB gene, allowing for a maximum of three consecutive wild-type nucleotides between each observed mutation. ( G ) The type of nucleotide substitutions identified within the ccdB gene, with or without the addition of 1 mM MnCl 2 . Each observed single-nucleotide deletion or insertion was counted as a one unique mutation.
Article Snippet: The resulting circular and randomly nicked DNA was purified by DNA-binding column and then added to the NSM Master Mix , containing a nucleotide mixture comprised of 2′-Deoxy-P-nucleoside-5′-O-triphosphate (dPTP) (Biolog Life Science Institute), 2′-Deoxyribavirin-5′-O-triphosphate (dRTP) (Biolog Life Science Institute), 2-Hydroxy-dATP (Jena Bioscience), and 8-Oxo-dGTP (Jena Bioscience), which was incubated at 55°C for 10–30 min. 250 Units/ml of T4 DNA ligase (Thermo Scientific), 10 mM DTT (Sigma–Aldrich) and 1 mM ATP (Thermo Scientific) was added to the NSM reaction, which was incubated for a further 60 min at 25°C, to ligate the DNA-backbone.
Techniques: Plasmid Preparation, Incubation, Introduce, Transformation Assay, Mutagenesis, Sequencing
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![NSM enables AT-content manipulation and homopolymeric mutations within random DNA regions spanning more than eight consecutive nucleotides. ( A ) The possible base-pairings of the synthetic nucleotides dPTP and dRTP, and the oxidized nucleotides 8-Oxo-dGTP and 2-Hydroxy-dATP [ – ]. Solid lines indicate strong base-pairing affinity, dashed lines indicate weak base-paring affinity. Illustration of the molecular structures of each mutagenic nucleotide compared to the conventional nucleotides: dATP, dCTP, dGTP, and dTTP. The oxidized nucleotide 2-Hydroxy-dATP exists in two primary tautomeric forms. Illustration created with BioRender.com . ( B ) The number of consecutive mutational changes that can be introduced by NSM during a 15 min nick-translation reaction using TLS DNAP, FEN1, 1 mM MnCl 2 , and a nucleotide mixture comprised of 20 µM of dPTP and 2-Hydroxy-dATP, and 10 µM of dRTP and 8-Oxo-dGTP. NSM-mutated pUC19 plasmids were sequenced by single-colony Sanger sequencing ( n = 91). The mutated region was defined as the distance (nt) between the first and last observed mutation in the ccdB gene, allowing for a maximum of three consecutive wild-type nucleotides between each observed mutation. ( C ) The type of nucleotide substitutions identified within the ccdB gene after a 15 min NSM-reaction (Fig. ). Each observed single-nucleotide deletion or insertion was counted as a one unique mutation. ( D ) The number of consecutive mutational changes that can be introduced by NSM during a 30 min nick-translation reaction using TLS DNAP, FEN1, 1 mM MnCl 2 , and 1 mM of only dPTP ( n = 34) or dRTP ( n = 33). NSM-mutated plasmids were sequenced by single-colony Sanger sequencing. The length of the mutated region was defined the same as in Figs and . ( E ) The type of nucleotide substitutions identified within the ccdB gene after a 30 min NSM reaction (Fig. ). Each observed single-nucleotide deletion or insertion was counted as a one unique mutation.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0153/pmc13010153/pmc13010153__gkag236fig7.jpg)