bfai  (New England Biolabs)

Journal: Journal of applied microbiology

Article Title: Isolation and basic characterization of a β-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture

doi: 10.1111/j.1365-2672.2009.04461.x

Figure Lengend Snippet: 16S ARDRA patterns on 2% Agarose. Digestion with Mse I ( A ) and Bfa I ( B ). Lanes 1 8: 50 bp ladder (A) , 100 bp ladder (B) . Lane 2: L. brevis DSMZ 20054. Lane 3: O. oeni DSMZ 20252. Lane 4: L. brevis SK3. Lane 5: Pediococcus acidilacti . Lane 6: O. oeni SK3Ba, Lane 7: O. oeni SK3Bb.

Article Snippet: Restriction analysis of the amplified 16S-rDNA fragment was done with the restriction enzymes Mse I and Bfa I (New England Biolabs) for 2 h at 37°C.

Techniques:

Journal: Breeding Science

Article Title: DNA marker development by the allele-specific detection of powdery mildew resistance loci derived from Japanese domestic tobacco cultivar ‘Kokubu’

doi: 10.1270/jsbbs.20011

Figure Lengend Snippet: Agarose gel electrophoresis profiles of NtMLO alleles detected using the cleaved amplified polymorphic sequence (CAPS) method. Amplified DNA fragments were digested by Bci T130I or Bfa I for NtMLO1 , and Bse RI for NtMLO2 . Bci T130I and Bfa I digests the wild-type (susceptible) and mutant (resistant) NtMLO1 fragments, respectively, and Bse RI digests mutant (resistant) NtMLO2 fragments. In the heterozygotes, three (digested and undigested) DNA fragments are generated by the restriction enzyme treatment. 1: ‘Tsukuba 1’ (resistant); 2: ‘K326’ (susceptible); 3: F 1 hybrid of ‘Tsukuba 1’ × ‘K326’ (susceptible); 4: ‘Kokubu’ (resistant); 5: ‘Ibusuki’ (susceptible); 6: F 1 hybrid of ‘Kokubu’ × ‘Ibusuki’ (susceptible).

Article Snippet: The following enzymes were selected for the digestion of the DNA: Bfa I (New England Biolabs) or Bci T130I (Takara) for NtMLO1 fragment digestion; Bse RI (New England Biolabs) for NtMLO2 fragment digestion.

Techniques: Agarose Gel Electrophoresis, Amplification, Sequencing, Mutagenesis, Generated

Journal: Breeding Science

Article Title: DNA marker development by the allele-specific detection of powdery mildew resistance loci derived from Japanese domestic tobacco cultivar ‘Kokubu’

doi: 10.1270/jsbbs.20011

Figure Lengend Snippet: Alignment of NtMLO genomic DNA sequences and restriction enzyme recognition sites used in the cleaved amplified polymorphic sequence (CAPS) method. The NtMLO1 gene of powdery mildew-resistant cultivars harbors two-base transitions (AG to TA) at the 3ʹ end of intron 7. In the NtMLO2 gene, the fourth and fifth nucleotide (AG) of intron 6 are deleted in powdery mildew-resistant cultivars. The CAPS method detects these polymorphisms by restriction enzyme digestion patterns. Bci T130I and Bfa I recognize and digest the wild-type (powdery mildew-susceptible) and mutant (powdery mildew-resistant) NtMLO1 genomic DNA sequence, respectively. Bse RI recognizes and digests the mutant (powdery mildew-resistant) NtMLO2 genomic DNA sequence. Intron sequences are shown in bold letters. Arrow heads indicate the digestion site of each enzyme.

Article Snippet: The following enzymes were selected for the digestion of the DNA: Bfa I (New England Biolabs) or Bci T130I (Takara) for NtMLO1 fragment digestion; Bse RI (New England Biolabs) for NtMLO2 fragment digestion.

Techniques: Amplification, Sequencing, Mutagenesis

Journal: American Journal of Human Genetics

Article Title: Mutations in FOXC2 (MFH-1), a Forkhead Family Transcription Factor, Are Responsible for the Hereditary Lymphedema-Distichiasis Syndrome

doi:

Figure Lengend Snippet: Mutation analysis in two families with hereditary lymphedema. Mutations in the FOXC2 gene were detected by direct sequencing of the PCR-amplified coding region of the gene. Results were confirmed by sequencing the reverse strand and by restriction-enzyme analysis. a, Sequencing revealed a TAC→TAG nonsense mutation (C297G) ( arrow, left panel ) in the proband (II-7) in family 1, creating a novel Bfa I restriction site. The right panel shows the cosegregation of the novel restriction site with the LD phenotype. Fetus II-6 was also shown to have the mutation, by restriction digest analysis (not shown). A 439-bp region of the FOXC2 gene was PCR amplified and digested with Bfa I. Products from normal alleles are not cut and remain 439 bp, whereas the mutant allele is cut into 383- and 53-bp fragments (not retained on gel). b, Sequence analysis revealing a 4-bp insertion in the proband (IV-2) in family 2. The GGCC insertion creates a shifted sequence beginning at the arrow ( left panel ). A novel Nae I restriction site is created by the insertion. The right panel shows the cosegregation of the Nae I fragments with the LD phenotype in family 2. PCR was used to amplify a 125-bp region of the FOXC2 gene (129 bp for the mutant allele). Nae I digestion of the PCR products results in 35- and 90-bp fragments for normal alleles, because of the presence of a naturally occurring Nae I site in the product. In alleles with the insertion, the 129-bp product is cut twice, generating two 35-bp fragments and a 59-bp fragment. Bands labeled “HD” show uncut heteroduplexes.

Article Snippet: PCR products were column purified (Qiagen) and were digested with Bfa I (New England Biolabs) at 37°C for 1 h prior to gel electrophoresis.

Techniques: Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification, Labeling

Journal: Nature Communications

Article Title: Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

doi: 10.1038/s41467-021-27004-1

Figure Lengend Snippet: Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.

Article Snippet: Then, 200 ng (2 µL) of annealed ssODNs was digested with either Bfa I or PvuII-HF (1 U, New England Biolabs) in CutSmart buffer (1×) in a total volume of 10 µL and incubated at 37 °C for 1 h. Immediately after digestion, reactions were separated on an agarose gel (1.5%) stained with SYBR Safe (1×), imaged (UVP BioDoc-It) and analysed semi-quantitatively by gel densitometry using ImageJ (Supplementary Data ).

Techniques: Sequencing, Transfection