t4pol  (New England Biolabs)


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    Structured Review

    New England Biolabs t4pol
    ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, <t>T4pol</t> or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .
    T4pol, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4pol/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4pol - by Bioz Stars, 2022-06
    97/100 stars

    Images

    1) Product Images from "ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes"

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1249

    ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .
    Figure Legend Snippet: ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .

    Techniques Used: Plasmid Preparation, Clone Assay

    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
    Figure Legend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Techniques Used: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

    2) Product Images from "ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes"

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1249

    ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .
    Figure Legend Snippet: ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .

    Techniques Used: Plasmid Preparation, Clone Assay

    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
    Figure Legend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Techniques Used: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

    3) Product Images from "ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes"

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1249

    ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .
    Figure Legend Snippet: ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .

    Techniques Used: Plasmid Preparation, Clone Assay

    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
    Figure Legend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Techniques Used: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

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    New England Biolabs t4 dna polymerase
    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized <t>T4</t> DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2022-06
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    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Labeling, Incubation, Negative Control

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation