t4 ligase  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    T4 DNA Polymerase 5 U µL
    Description:
    Thermo Scientific T4 DNA Polymerase is a template dependent DNA polymerase that catalyzes 5 3 synthesis from primed single stranded DNA The enzyme has a 3 5 exonuclease activity but lacks 5 3 exonuclease activity Highlights• Stronger 3 5 exonuclease activity on single stranded than on double stranded DNA and greater more than 200 times than DNA polymerase I E coli and Klenow fragment• Active in Thermo Scientific restriction enzyme PCR RT and T4 DNA Ligase buffersApplications• Blunting of DNA ends fill in of 5 overhangs or and removal of 3 overhangs see​ References1 2 • Blunting of PCR products with 3 dA overhangs• Synthesis of labeled DNA probes by the replacement reaction see​ Reference3 • Oligonucleotide directed site specific mutagenesis see​ Reference4 • Ligation independent cloning of PCR products
    Catalog Number:
    ep0061
    Price:
    None
    Applications:
    Cloning|PCR Cloning|Restriction Enzyme Cloning|Mutagenesis
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher t4 ligase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    Thermo Scientific T4 DNA Polymerase is a template dependent DNA polymerase that catalyzes 5 3 synthesis from primed single stranded DNA The enzyme has a 3 5 exonuclease activity but lacks 5 3 exonuclease activity Highlights• Stronger 3 5 exonuclease activity on single stranded than on double stranded DNA and greater more than 200 times than DNA polymerase I E coli and Klenow fragment• Active in Thermo Scientific restriction enzyme PCR RT and T4 DNA Ligase buffersApplications• Blunting of DNA ends fill in of 5 overhangs or and removal of 3 overhangs see​ References1 2 • Blunting of PCR products with 3 dA overhangs• Synthesis of labeled DNA probes by the replacement reaction see​ Reference3 • Oligonucleotide directed site specific mutagenesis see​ Reference4 • Ligation independent cloning of PCR products
    https://www.bioz.com/result/t4 ligase/product/Thermo Fisher
    Average 99 stars, based on 527 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "A Rapid Cloning Method Employing Orthogonal End Protection"

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037617

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    Figure Legend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Techniques Used: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    2) Product Images from "Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs"

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076117

    Summary of the Golden GATEway cloning kit. A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway TM  entry vectors at any position (Threeway Gateway TM  cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).
    Figure Legend Snippet: Summary of the Golden GATEway cloning kit. A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway TM entry vectors at any position (Threeway Gateway TM cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Ligation

    3) Product Images from "Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons"

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons

    Journal: Journal of neurochemistry

    doi:

    Positive and negative controls for chromosome conformation capture. All possible restriction fragments after  Bgl II  digestions were present in equimolar amounts. A. Two regions of calreticulin gene,  CalR2.8  and  CalR4.4 , were cross-linked and represented a positive control. On the other hand,  COX4i1  and  CalR  were two functionally unrelated genes, and they did not interact in our 3C reactions. B.  CalR2.8  and  CalR4.4 , as well as  COX4i1  or  COX8a  and  Grin1 ,  Gria2 , or  Nos1 , respectively, did not interact in the absence of a cross-linking agent (formaldehyde) or T4 ligase. Experiments were performed in triplicates.
    Figure Legend Snippet: Positive and negative controls for chromosome conformation capture. All possible restriction fragments after Bgl II digestions were present in equimolar amounts. A. Two regions of calreticulin gene, CalR2.8 and CalR4.4 , were cross-linked and represented a positive control. On the other hand, COX4i1 and CalR were two functionally unrelated genes, and they did not interact in our 3C reactions. B. CalR2.8 and CalR4.4 , as well as COX4i1 or COX8a and Grin1 , Gria2 , or Nos1 , respectively, did not interact in the absence of a cross-linking agent (formaldehyde) or T4 ligase. Experiments were performed in triplicates.

    Techniques Used: Positive Control

    4) Product Images from "DNAzyme-based probe for circulating microRNA detection in peripheral blood"

    Article Title: DNAzyme-based probe for circulating microRNA detection in peripheral blood

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S89560

    The schematic strategy for miRNA detection. Notes: A DNAzyme-based probe contains three domains: a miRNA-binding domain (the bubble structure), a DNAzyme domain, and a substrate domain. The binding of miRNA to DNAzyme-based probe initiates T4 ligase reaction. ( A ) Secondary structure of the incomplete miRNA-specific DNAzyme. ( B ) The intact miRNA-specific DNAzyme cleaved into two fragments in the presence of Cu 2+ . ( C ) The cleaved product is then applied to formation of self-assembled DNA concatemers, eventually resulting amplified fluorescent signals in the presence of SYBR Green I. Abbreviations: miRNA, microRNA; SA-MBs, streptavidin-coated microsphere beads.
    Figure Legend Snippet: The schematic strategy for miRNA detection. Notes: A DNAzyme-based probe contains three domains: a miRNA-binding domain (the bubble structure), a DNAzyme domain, and a substrate domain. The binding of miRNA to DNAzyme-based probe initiates T4 ligase reaction. ( A ) Secondary structure of the incomplete miRNA-specific DNAzyme. ( B ) The intact miRNA-specific DNAzyme cleaved into two fragments in the presence of Cu 2+ . ( C ) The cleaved product is then applied to formation of self-assembled DNA concatemers, eventually resulting amplified fluorescent signals in the presence of SYBR Green I. Abbreviations: miRNA, microRNA; SA-MBs, streptavidin-coated microsphere beads.

    Techniques Used: Binding Assay, Amplification, SYBR Green Assay

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice
    Article Snippet: .. The PCR products were ligated into the Bam HI/ Not I site of pPK-CMV-F4 (PromoCell GmbH, Heidelberg, Germany) mammalian expression vector using T4 ligase (Invitrogen, Carlsbad, CA, USA). .. All plasmids containing correct inserts (as confirmed by DNA sequencing) were propagated in Escherichia coli NOVA XG cells, amplified, extracted and purified using conventional methods.

    Incubation:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

    Expressing:

    Article Title: Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice
    Article Snippet: .. The PCR products were ligated into the Bam HI/ Not I site of pPK-CMV-F4 (PromoCell GmbH, Heidelberg, Germany) mammalian expression vector using T4 ligase (Invitrogen, Carlsbad, CA, USA). .. All plasmids containing correct inserts (as confirmed by DNA sequencing) were propagated in Escherichia coli NOVA XG cells, amplified, extracted and purified using conventional methods.

    Plasmid Preparation:

    Article Title: Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice
    Article Snippet: .. The PCR products were ligated into the Bam HI/ Not I site of pPK-CMV-F4 (PromoCell GmbH, Heidelberg, Germany) mammalian expression vector using T4 ligase (Invitrogen, Carlsbad, CA, USA). .. All plasmids containing correct inserts (as confirmed by DNA sequencing) were propagated in Escherichia coli NOVA XG cells, amplified, extracted and purified using conventional methods.

    Ligation:

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs
    Article Snippet: .. The 10nM annealed double-stranded oligo dilution was used as an insert in a standard ligation reaction with T4 ligase (5U, Thermo, Fisher) .. Golden Gate Protocol Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O.

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher t4 dna ligase
    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using <t>T4</t> DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 550 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher t4 rna ligase
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/Thermo Fisher
    Average 99 stars, based on 61 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Journal: Chembiochem

    Article Title: Practical Synthesis of Cap‐4 RNA

    doi: 10.1002/cbic.201900590

    Figure Lengend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Article Snippet: The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. .

    Techniques: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    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.

    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: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: DNA Ligation, Ligation, Staining, Marker

    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.

    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: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

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

    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.

    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: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: Ligation, Sequencing, Ancient DNA Assay

    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

    doi: 10.1016/j.bpj.2018.08.013

    Figure Lengend Snippet: DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Article Snippet: For ligation, T4 DNA ligase was used in standard conditions (25°C, 10 mM MgCl2 ), and the GC content of the target site was ∼50%.

    Techniques: Microscopy, Labeling, Ligation, Binding Assay, Imaging

    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Journal: RNA Biology

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2

    doi: 10.1080/15476286.2018.1518854

    Figure Lengend Snippet: Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Article Snippet: RNA was resuspended in 14.5 µl H2 O and subjected to T4 RNA Ligase reaction by adding 1 µl of 20 µM 5′-phosporylated RL3 (5′-P -GUGUCAGUCACUUCCAGCGG-Puromycin-3′), 2 µl 10× T4 Ligase Buffer, 2 µl BSA, 0.5 µl T4 RNA Ligase (ThermoFisher), and incubated overnight at 16°C.

    Techniques: Allele-specific Oligonucleotide, Selection, In Vivo, Generated, Sequencing, Combined Bisulfite Restriction Analysis Assay, Activity Assay, shRNA, Protein Concentration, Size-exclusion Chromatography, Ligation, Staining, Purification, Sample Prep, Labeling, Autoradiography