Journal: Nucleic Acids Research

Article Title: Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions

doi:

Figure Lengend Snippet: Time-dependent ligation reactions. The optimum reaction conditions were used, which are 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , 10 µM ATP, 20% DMSO, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol C4 template and 3 U T4 DNA ligase at 22°C. The graph shows the plots and fitted curves of each of the four ligation reactions analyzed (see inset for oligonucleotide sequences). The observed rate constants ( k obs ) for each reaction are given on the graph. The data points are the average of two independent assays.

Article Snippet: All ligation reactions consist of 3.125 pmol template, 2.5 pmol non-radiolabeled oligonucleotide (the acceptor molecule), 70 fmol radiolabeled oligonucleotide (the donor molecule) and 3 U T4 DNA ligase (Promega, Madison, WI).

Techniques: Ligation

Journal: Nucleic Acids Research

Article Title: Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions

doi:

Figure Lengend Snippet: Optimization of ligation reactions. ( A and B ) ATP and MgCl 2 concentration-dependent ligations using (c + p c)/T2. The MgCl 2 concentration was first optimized under standard reaction conditions of 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 1 mM ATP, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol template and 3 U T4 DNA ligase at 30°C for 18 h. An optimum concentration of 3 mM MgCl 2 was found, which was then used in the subsequent ATP-dependent assay. ( C ) DMSO-dependent ligation reaction using (c + p c)/C2 in 3 mM MgCl 2 and 10 µM ATP. ( D ) Time-dependent ligation reaction using (c + p c)/T2 in 3 mM MgCl 2 , 10 µM ATP and 20% DMSO. In each case, product is represented by squares and intermediate by circles.

Article Snippet: All ligation reactions consist of 3.125 pmol template, 2.5 pmol non-radiolabeled oligonucleotide (the acceptor molecule), 70 fmol radiolabeled oligonucleotide (the donor molecule) and 3 U T4 DNA ligase (Promega, Madison, WI).

Techniques: Ligation, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I

doi:

Figure Lengend Snippet: T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16 multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.

Article Snippet: T4 DNA-ligase was from Boehringer Mannheim.

Techniques: Activity Assay, Incubation, Inhibition, Electrophoresis, Autoradiography

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

doi: 10.1093/dnares/dsy037

Figure Lengend Snippet: 3′ Branch ligation by T4 DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.

Article Snippet: The gap ligation of AdB (ON28 and ON29) to the tagmented DNA was performed at 25°C for 1 h in reactions containing 100 pmol of the adapter, 600 U of T4 DNA ligase (Enzymatics, Inc.), and 3 ′ BL buffer.

Techniques: Ligation

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

doi: 10.1093/dnares/dsy037

Figure Lengend Snippet: 3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.

Article Snippet: The gap ligation of AdB (ON28 and ON29) to the tagmented DNA was performed at 25°C for 1 h in reactions containing 100 pmol of the adapter, 600 U of T4 DNA ligase (Enzymatics, Inc.), and 3 ′ BL buffer.

Techniques: Ligation