Journal: BMC Biochemistry

Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

doi: 10.1186/1471-2091-12-47

Figure Lengend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

Article Snippet: Bst DNA Polymerase large fragment exo-, pUC19 DNA, BsaI, NcoI and BspHI, T4 DNA ligase REase were from New England Biolabs (Ipswich, MA, USA).

Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling

Journal: BMC Biochemistry

Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

doi: 10.1186/1471-2091-12-47

Figure Lengend Snippet: Assessment of various DNA polymerases for their ability to incorporate BrdU . Complete and incomplete specific incorporation reactions (Figure 1) were carried out with 5 DNA Polymerases: Bst exo - (thermophilic), T4 (mesophilic), Taq (thermophilic), OptiTaq (thermophilic blend) and Pfu (hyperthermophilic) in the presence of BrdUTP. Lanes M, Perfect 100 bp Ladder; lane 1, PCR 1 fragment (379 bp); lane 2, BsaI-cleaved PCR 1 fragment; lane 3, PCR 2 fragment (625 bp); lane 4, BsaI-cleaved PCR 2 fragment; lane 5, BsaI restriction fragments: I (363 bp) and III (609 bp). Lanes 6-18 reactions with specified DNA Polymerases: lane 6, restriction fragments: I and III, T4; lane 7, restriction fragments: I and III, Bst exo - ; lane 8, restriction fragments: I and III, Bst exo - , T4 DNA Ligase; lane 9, restriction fragments: I and III, T4; lane 10, restriction fragments: I and III, T4, T4 DNA Ligase; lane 11, restriction fragments: I and III, Taq; lane 12, restriction fragments: I and III, Taq, T4 DNA Ligase; lane 13, restriction fragments: I and III, OptiTaq; lane 14, restriction fragments: I and III, OptiTaq, T4 DNA Ligase; lane 15, restriction fragments: I and III, Tfl; lane 16, restriction fragments: I and III, Tfl, T4 DNA Ligase; lane 17, restriction fragments: I and III, Pfu; lane 18, restriction fragments: I and III, Pfu, T4 DNA Ligase. I, III BsaI restriction fragments numbered as in Figure 1.

Article Snippet: Bst DNA Polymerase large fragment exo-, pUC19 DNA, BsaI, NcoI and BspHI, T4 DNA ligase REase were from New England Biolabs (Ipswich, MA, USA).

Techniques: Polymerase Chain Reaction

Journal: BMC Biochemistry

Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

doi: 10.1186/1471-2091-12-47

Figure Lengend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 441 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked); lane 1, 260 bp BsaI-cleaved PCR (restriction fragment I); lane 2, 208 bp BsaI-cleaved PCR (restriction fragment III); lane 3, BrdUTP-filled restriction fragments I and III, T4 DNA ligase; lane 4, BrdUTP-filled restriction fragments I and III; lane 5, dTTP-filled restriction fragment I and BrdUTP-filled restriction fragment III, T4 DNA ligase; lane 6, dTTP-filled restriction fragment I and BrdU-filled restriction fragment III. Lanes 7-9, controls of enzymes functional purity: lane 7, control PCR fragment with internal BsaI site; lane 8, BsaI-cleaved control PCR fragment; lane 9, BsaI-cleaved control PCR fragment after addition of T4 DNA Ligase; lane M, Perfect 100 bp Ladder. I, III BsaI restriction fragments numbered as in Figure 1.

Article Snippet: Bst DNA Polymerase large fragment exo-, pUC19 DNA, BsaI, NcoI and BspHI, T4 DNA ligase REase were from New England Biolabs (Ipswich, MA, USA).

Techniques: Polymerase Chain Reaction, Functional Assay

Journal: BMC Biotechnology

Article Title: GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system

doi: 10.1186/s12896-020-00616-z

Figure Lengend Snippet: Efficiency of different BsaI enzymes. To test the efficiency of the BsaI-HFv2 enzyme, the same 15 entry vectors were used in a Golden Gate reaction with either the standard BsaI enzyme or BsaI-HFv2. Ten clones resulting from each assembly reaction were picked, digested with EcoRV, and analyzed via agarose gel electrophoresis. A schematic of the DNA sizing ladder and the predicted band pattern for the assembly reaction is shown to the left of the respective agarose gel. Clones demonstrating correct assembly based on the pattern of bands are marked with a green asterisk. While the reaction performed with the standard BsaI enzyme resulted in no correct clones, the reaction with the BsaI-HFv2 enzyme showed 9/10 correct clones

Article Snippet: During another especially challenging project where 15 expression cassettes were to be assembled, we compared the influence on efficiency of two BsaI enzyme variants (BsaI from New England Biolabs Cat. No. R0535S and BsaI-HFv2 also from NEB Cat. No. R3733S).

Techniques: Clone Assay, Agarose Gel Electrophoresis

Journal: BMC Microbiology

Article Title: Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history

doi: 10.1186/1471-2180-8-81

Figure Lengend Snippet: Chromatograms of PCR-RFLP assays and sequencing for detection of nucleotide alterations of 23S rRNA . H. Pylori 26695 and CLR r -1 were used as negative and positive control of A2143G mutation. BsaI digestion of the PCR products of representative samples was displayed on 8% PAGE gel. The 289 bp A2143G-positive PCR products were cleaved into a 199 bp and a 90 bp fragments ( A ). The A2143G mutation was also confirmed by sequencing of the PCR products of 23S rRNA ( B , displayed 2140–2154 fragment). H. Pylori 26695 and a 2142G clone were used as negative and positive control of A2142G mutation. MboII digestion of the PCR products of representative samples was displayed on 2% agarose gel. The 289 bp A2142G-positive PCR products of 2142G were cleaved into an 182 bp and a 107 bp fragments. The PCR product of GJ2040 was cleaved into a 164 bp and a 125 bp fragments; and the product of GJ2111 was cleaved into 245 bp and 44 bp fragment(s) ( C ). The A2142G and other mutations were confirmed by sequencing ( D ). Two new MboII -sensitive sequences were characterized as CTTCA (2222–2226) for GJ2040 and GAAG (2081–2084) for GJ2111.

Article Snippet: RFLP assays The 289 bp amplicon of 23S rRNA was digested with the restriction enzymes BsaI and MboII (New England Biolabs, USA) in order to detect A2143G and A2142G point mutations, respectively (Fig ) [ , ].

Techniques: Polymerase Chain Reaction, Sequencing, Positive Control, Mutagenesis, Polyacrylamide Gel Electrophoresis, Agarose Gel Electrophoresis

Journal: Journal of Korean Medical Science

Article Title: Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea

doi: 10.3346/jkms.2014.29.9.1240

Figure Lengend Snippet: Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

Article Snippet: Amplicons (424 bp each) of the 23S rRNA gene were digested with either Bsa I (New England BioLabs, Beverly, MA, USA) for 14 hr at 50℃ or Bbs I (New England BioLabs) for 14 hr at 37℃ to detect the A2144G and A2143G mutations, respectively ( ).

Techniques: Mutagenesis, DNA Sequencing

Journal: bioRxiv

Article Title: High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

doi: 10.1101/2020.05.12.091991

Figure Lengend Snippet: Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.

Article Snippet: For controls, target plasmids and empty pUC19 were linearized by restriction enzyme digestion using BsaI-HF and nicked using a nicking enzyme Nt.BspQI (New England Biolabs).

Techniques: Plasmid Preparation, Incubation

Journal: bioRxiv

Article Title: High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

doi: 10.1101/2020.05.12.091991

Figure Lengend Snippet: High-throughput in vitro analysis of Cas9 mismatch tolerance. (A) Outline and workflow of the high-throughput in vitro cleavage assay. (B) Representative agarose gel showing time course cleavage of negatively supercoiled (nSC) plasmid containing a fully matched PS4 target (pTarget PS4, left) and plasmid library PS4 (pLibrary PS4, right) by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); (-r) = pTarget or pLibrary incubated with Cas9 only at 37 °C for the longest time point in the assay (3 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (C) Overall cleavage of the pLibrary PS4 by Cas9 indicating the decrease in supercoiled (nSC) pool and appearance of nicked (n) and linear (li) pools over time. The 0 time point is the quantification of the negative control pLibrary (i.e. pLibrary run on a gel after preparation as represented in Fig. S2A). Values plotted represent an average of two replicates. Error bars are SEM.

Article Snippet: For controls, target plasmids and empty pUC19 were linearized by restriction enzyme digestion using BsaI-HF and nicked using a nicking enzyme Nt.BspQI (New England Biolabs).

Techniques: High Throughput Screening Assay, In Vitro, Cleavage Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Negative Control

Journal: Jundishapur Journal of Microbiology

Article Title: Detection of 23SrRNA Mutations Strongly Related to Clarithromycin Resistance in Helicobacter pylori Strains Isolated From Patients in the North of Iran

doi: 10.5812/jjm.29694

Figure Lengend Snippet: Gel Electrophoresis of Digested 429 bp Polymerase Chain Reaction Product of 23S rRNA Gene With BsaI and MboII Enzymes Lane 1, DNA ladder (100 bp); Lane 2, clarithromycin resistant strain digested with BsaI at two sites; Lane 3 and 4, wild types of H. pylori (susceptible to clarithromycin) digested with BsaI, with one digestion site; Lane 5, wild type (susceptible to clarithromycin) treated with MboII with no digestion site.

Article Snippet: To detect the A-G point mutations at positions 2142 and 2143, the PCR products were digested with the MboII and BsaI (MBI Fermentas, Lithuania) restriction enzymes, respectively, according to the manufacturer’s instructions, and analyzed on 2% agarose gel containing Sybrsafe.

Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction