Journal: Nucleic Acids Research

Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

doi: 10.1093/nar/gkx033

Figure Lengend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

Article Snippet: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

Techniques: DNA Ligation, Ligation, Staining, Marker

Journal: Nucleic Acids Research

Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

doi: 10.1093/nar/gkx033

Figure Lengend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

Article Snippet: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

Techniques: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

Journal: Nucleic Acids Research

Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

doi: 10.1093/nar/gkx033

Figure Lengend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

Article Snippet: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

Techniques: Ligation, Sequencing, Ancient DNA Assay

Journal: Nucleic Acids Research

Article Title: The Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli

doi: 10.1093/nar/gkm722

Figure Lengend Snippet: Comparison of cleavage patterns of oligonucleotide containing the 5′-dCdTdCdTdTdC-3′ corresponding to a T4 DNA sequence by wild-type and S176N mutant forms of Endo IV. A 45-base oligonucleotide (Cy5–T4A) based on the sequence of T4 DNA and labeled at its 5′ end with Cy5 was used as the substrate (10 μM) for assay of the activity of wild-type (0.5, 1.0, 2.0 or 4.0 μg/ml) ( A ) or S176N mutant (1.0, 2.0, 4.0 or 8.0 μg/ml) ( B ) forms of Endo IV. The reaction products were separated by electrophoresis on a 10% polyacrylamide gel containing 7 M urea and were visualized with an image analyzer. Lane (−) represents a reaction mixture incubated in the absence of enzyme. Lane M represents a mixture of oligonucleotides labeled at their 5′ ends with Cy5 and with sequences identical to those of residues 1 to 19, 1 to 21 or 1 to 24 of the substrate. Cleavage sites of the substrate are indicated by arrows, and dC sites in dG 19 –dC 20 –dT 21 , dT 21 –dC 22 –dT 23 and dT 24 –dC 25 –dA 26 are indicated by a, b and c, which correspond to the dC 1 , dC 3 and dC 6 sites of the 5′-dC 1 dT 2 dC 3 dT 4 dT 5 dC 6 -3′ tract, respectively.

Article Snippet: Cleavage analysis of an oligonucleotide corresponding to a T4 DNA sequence by Endo IV Hydrolysis of a 45-base oligonucleotide (Cy5–T4A, see Materials section) based on the sequence of T4 DNA and labeled at its 5′ end with Cy5 (Sigma-Aldrich) was performed as described above for the Endo IV assay at a substrate concentration of 10 μM and enzyme concentrations of either 0.5–4 μg/ml for wild-type Endo IV or 1–8 μg/ml for Endo IV(S176N).

Techniques: Sequencing, Mutagenesis, Labeling, Activity Assay, Electrophoresis, Incubation

Journal: Journal of Virology

Article Title: Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

doi: 10.1128/JVI.75.22.10573-10581.2001

Figure Lengend Snippet: Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

Article Snippet: Reactions were carried out at 37°C for 30 min in a final volume of 20 μl containing 400 ng of TNAs, 100 μM each dNTP, and 2 U of T4 DNA polymerase (Boehringer Mannheim) in the incubation buffer supplied, then the concentration of each dNTP was brought to 200 μM, and incubation was prolonged for another 30 min.

Techniques: Incubation, Transgenic Assay, Infection, Migration