dna repair  (New England Biolabs)


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    New England Biolabs dna repair
    Genome-wide base composition bias curves in Illumina reads from PCR-free human <t>DNA</t> libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or <t>NEBNext</t> Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Dna Repair, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    2) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    3) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    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 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( 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.
    Figure Legend 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 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( 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.

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

    4) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    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.
    Figure Legend 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.

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

    5) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    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.
    Figure Legend 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.

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

    6) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    Related Articles

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    Article Snippet: .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France). .. The end-repaired products were then purified using 1.0 x volume of AMPure XP beads (Beckman-Coulter) and eluted in 25μL of nuclease-free.

    Amplification:

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    Ligation:

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    Article Snippet: .. DNA Library Preparation Libraries for next-generation sequencing were built for all three DNA extracts using a modified version of ref. as outlined in ref. , where blunt end repair was performed using NEBNext End-Repair (New England Biolabs Inc.) and Bst was inactivated by heat (20 minutes at 80 °C). .. Thomas Kent’s DNA library was prepared in a dedicated ancient DNA lab whereas the libraries for the DNA of two modern relatives were prepared in a modern DNA lab in UCD Earth Institute’s Area 52.

    Purification:

    Article Title: Multiplexed Nanopore Sequencing of HLA-B Locus in Māori and Pacific Island Samples
    Article Snippet: .. Briefly, 5 μg of purified amplicon library was prepared with the NEBNext end repair module (New England Biolabs), then dA-tailed using the NEBNext dA-tailing module (New England Biolabs). .. The final prepared library from 49 participants was loaded into the MinION R9.4 flowcell (Oxford Nanopore Technologies).

    Real-time Polymerase Chain Reaction:

    Article Title: Architectural proteins and pluripotency factors cooperate to orchestrate the transcriptional response of hESCs to temperature stress
    Article Snippet: .. Fragment ends were repaired using the NEBNext End Repair Module and adenosine was added at the 3’ ends of fragments using Klenow fragment (3’ to 5’ exo minus, New England Biolabs), universal adaptors were ligated to the A-tailed DNA fragments at room temperature for 1 h with T4 DNA ligase (New England Biolabs) and amplified with Illumina barcoded primers using KAPA SYBR FAST qPCR Master Mix for 5~12 PCR cycles to obtain enough DNA for sequencing. .. Generated libraries were paired-end sequenced on Illumina HiSeq2500 v4.

    Modification:

    Article Title: The Identification of a 1916 Irish Rebel: New Approach for Estimating Relatedness From Low Coverage Homozygous Genomes
    Article Snippet: .. DNA Library Preparation Libraries for next-generation sequencing were built for all three DNA extracts using a modified version of ref. as outlined in ref. , where blunt end repair was performed using NEBNext End-Repair (New England Biolabs Inc.) and Bst was inactivated by heat (20 minutes at 80 °C). .. Thomas Kent’s DNA library was prepared in a dedicated ancient DNA lab whereas the libraries for the DNA of two modern relatives were prepared in a modern DNA lab in UCD Earth Institute’s Area 52.

    Polymerase Chain Reaction:

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France). .. The end-repaired products were then purified using 1.0 x volume of AMPure XP beads (Beckman-Coulter) and eluted in 25μL of nuclease-free.

    Article Title: Architectural proteins and pluripotency factors cooperate to orchestrate the transcriptional response of hESCs to temperature stress
    Article Snippet: .. Fragment ends were repaired using the NEBNext End Repair Module and adenosine was added at the 3’ ends of fragments using Klenow fragment (3’ to 5’ exo minus, New England Biolabs), universal adaptors were ligated to the A-tailed DNA fragments at room temperature for 1 h with T4 DNA ligase (New England Biolabs) and amplified with Illumina barcoded primers using KAPA SYBR FAST qPCR Master Mix for 5~12 PCR cycles to obtain enough DNA for sequencing. .. Generated libraries were paired-end sequenced on Illumina HiSeq2500 v4.

    Article Title: RNA structure replaces the need for U2AF2 in splicing
    Article Snippet: .. To build a sequenceable library, the resuspended RNA was (1) reverse-transcribed into cDNA by SuperScript II (Invitrogen) and random 9-mers, (2) made into double-stranded cDNA with the NEBNext mRNA Second Strand Synthesis Module (NEB), (3) made into blunt-end dsDNA with the NEBNext End Repair Module (NEB), (4) made into dsDNA with a single A overhang with NEBNext dA-Tailing Module (NEB), (5) ligated with Illumina sequencing adaptors with NEBNext Quick Ligation Module (NEB), and (6) amplified to meet the sequencing requirement by NEBNext High-Fidelity 2× PCR Master Mix (NEB) and two outer primers recognizing the ligated Illumina adaptors (5′-AATGATACGGCGACCACCGAGATCTACAC and 5′-CAAGCAGAAGACGGCATACGAGAT). ..

    Sequencing:

    Article Title: Architectural proteins and pluripotency factors cooperate to orchestrate the transcriptional response of hESCs to temperature stress
    Article Snippet: .. Fragment ends were repaired using the NEBNext End Repair Module and adenosine was added at the 3’ ends of fragments using Klenow fragment (3’ to 5’ exo minus, New England Biolabs), universal adaptors were ligated to the A-tailed DNA fragments at room temperature for 1 h with T4 DNA ligase (New England Biolabs) and amplified with Illumina barcoded primers using KAPA SYBR FAST qPCR Master Mix for 5~12 PCR cycles to obtain enough DNA for sequencing. .. Generated libraries were paired-end sequenced on Illumina HiSeq2500 v4.

    Article Title: RNA structure replaces the need for U2AF2 in splicing
    Article Snippet: .. To build a sequenceable library, the resuspended RNA was (1) reverse-transcribed into cDNA by SuperScript II (Invitrogen) and random 9-mers, (2) made into double-stranded cDNA with the NEBNext mRNA Second Strand Synthesis Module (NEB), (3) made into blunt-end dsDNA with the NEBNext End Repair Module (NEB), (4) made into dsDNA with a single A overhang with NEBNext dA-Tailing Module (NEB), (5) ligated with Illumina sequencing adaptors with NEBNext Quick Ligation Module (NEB), and (6) amplified to meet the sequencing requirement by NEBNext High-Fidelity 2× PCR Master Mix (NEB) and two outer primers recognizing the ligated Illumina adaptors (5′-AATGATACGGCGACCACCGAGATCTACAC and 5′-CAAGCAGAAGACGGCATACGAGAT). ..

    Nanopore Sequencing:

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France). .. The end-repaired products were then purified using 1.0 x volume of AMPure XP beads (Beckman-Coulter) and eluted in 25μL of nuclease-free.

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    New England Biolabs rna dependent dna recombinational repair rad52 dependent rna bridging reactions
    <t>RAD52</t> promotes <t>RNA-dependent</t> <t>DNA</t> recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated
    Rna Dependent Dna Recombinational Repair Rad52 Dependent Rna Bridging Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna dependent dna recombinational repair rad52 dependent rna bridging reactions/product/New England Biolabs
    Average 92 stars, based on 1 article reviews
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    90
    New England Biolabs dna repair
    Genome-wide base composition bias curves in Illumina reads from PCR-free human <t>DNA</t> libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or <t>NEBNext</t> Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Dna Repair, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna repair/product/New England Biolabs
    Average 90 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    dna repair - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

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    RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinant, Recombinase Polymerase Amplification

    RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinase Polymerase Amplification, Incubation, Ligation, DNA Ligation

    RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinase Polymerase Amplification, Sequencing

    Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Ligation

    RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinant

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    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: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Article Snippet: FFPE kidney tumor genomic DNA (BioChain Institute Inc., Newark, CA) libraries were prepared with or without DNA repair following NEBNext Ultra II DNA library or immobilized enzyme workflows as described above.

    Techniques: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    The repair of compatible ends required DNA-PK but not ATM. ( A ) The pMBPII plasmid was digested by XhoI to generate compatible DSB ends. The DNA substrate was incubated in Xenopus egg extracts, re-isolated, and transformed into bacteria cells. The final repair products were isolated from bacteria colonies and subjected to sequencing analysis. The repair product with large-fragment deletion (200 bp-2 kb) was determined by agarose gel electrophoresis (data not shown). ( B ) The repair assay was established as in panel A. The extract was supplemented with or without DNA-PKcs inhibitor NU7441. The repair activity (in relative to the control extract) was measured by colony numbers. ( C ) The repair assay was established as in panel A. The extract was supplemented with or without ATM inhibitor KU60019. The repair activity (in relative to the control extract) was measured by colony numbers. ( D ) The specificity of ATM and DNA-PKcs inhibitors were confirmed by immunoblotting. Xenopus egg extract were treated with double-stranded oligonucleotides and specific inhibitors as indicated. After incubation for 30 min at room temperature, samples were analyzed using specific antibodies as indicated. In panels B C, a minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Journal: Scientific Reports

    Article Title: Non-homologous end joining repair in Xenopus egg extract

    doi: 10.1038/srep27797

    Figure Lengend Snippet: The repair of compatible ends required DNA-PK but not ATM. ( A ) The pMBPII plasmid was digested by XhoI to generate compatible DSB ends. The DNA substrate was incubated in Xenopus egg extracts, re-isolated, and transformed into bacteria cells. The final repair products were isolated from bacteria colonies and subjected to sequencing analysis. The repair product with large-fragment deletion (200 bp-2 kb) was determined by agarose gel electrophoresis (data not shown). ( B ) The repair assay was established as in panel A. The extract was supplemented with or without DNA-PKcs inhibitor NU7441. The repair activity (in relative to the control extract) was measured by colony numbers. ( C ) The repair assay was established as in panel A. The extract was supplemented with or without ATM inhibitor KU60019. The repair activity (in relative to the control extract) was measured by colony numbers. ( D ) The specificity of ATM and DNA-PKcs inhibitors were confirmed by immunoblotting. Xenopus egg extract were treated with double-stranded oligonucleotides and specific inhibitors as indicated. After incubation for 30 min at room temperature, samples were analyzed using specific antibodies as indicated. In panels B C, a minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Article Snippet: DNA repair assay in Xenopus egg extracts Plasmid pMBP-parallel2 (pMBPII) was digested with different restriction enzymes (New England Biolabs, MA) to generate linearized DNA with various end structures.

    Techniques: Plasmid Preparation, Incubation, Isolation, Transformation Assay, Sequencing, Agarose Gel Electrophoresis, Activity Assay

    Processing of non-compatible ends is flexible and specific to the structure of DSB ends. Various NHEJ repair templates with non-compatible ends, as in Fig. S2 , were incubated in Xenopus egg extracts, re-isolated, and transformed into bacteria cells. Final repair products were isolated and subjected to sequencing analysis. The NHEJ templates include: blunt/3′-overhang ( A ), blunt with 5′-overhang ( B ), 3′-overhang/5′-overhang (C ), 3′-overhang/3′-overhang ( D ), and 5′-overhang/5′-overhang ( E ). The repair assay was performed with or without ATM inhibitor as in Fig. 3B . In each reaction, approximately 15–20 final repair products were sequenced and shown. Nucleotides deleted during DNA repair were indicated by empty triangles. Repair products with large-fragment deletion (200 bp-2 kb) were determined by agarose gel electrophoresis (data not shown).

    Journal: Scientific Reports

    Article Title: Non-homologous end joining repair in Xenopus egg extract

    doi: 10.1038/srep27797

    Figure Lengend Snippet: Processing of non-compatible ends is flexible and specific to the structure of DSB ends. Various NHEJ repair templates with non-compatible ends, as in Fig. S2 , were incubated in Xenopus egg extracts, re-isolated, and transformed into bacteria cells. Final repair products were isolated and subjected to sequencing analysis. The NHEJ templates include: blunt/3′-overhang ( A ), blunt with 5′-overhang ( B ), 3′-overhang/5′-overhang (C ), 3′-overhang/3′-overhang ( D ), and 5′-overhang/5′-overhang ( E ). The repair assay was performed with or without ATM inhibitor as in Fig. 3B . In each reaction, approximately 15–20 final repair products were sequenced and shown. Nucleotides deleted during DNA repair were indicated by empty triangles. Repair products with large-fragment deletion (200 bp-2 kb) were determined by agarose gel electrophoresis (data not shown).

    Article Snippet: DNA repair assay in Xenopus egg extracts Plasmid pMBP-parallel2 (pMBPII) was digested with different restriction enzymes (New England Biolabs, MA) to generate linearized DNA with various end structures.

    Techniques: Non-Homologous End Joining, Incubation, Isolation, Transformation Assay, Sequencing, Agarose Gel Electrophoresis

    Repair of DSB substrates in Xenopus egg extract. ( A ) Schematic diagram of the DSB repair assay. As described in Materials and Methods, linearized plasmid DNA was generated and incubated in Xenopus egg extract. Plasmid DNA was then re-isolated from egg extracts, and transformed into bacteria cells. The final repair products were isolated from bacteria colonies and subjected to sequencing analysis. ( B ) As in panel A, incubation of plasmid DNA linearized with Xho1 and Kpn1 in Xenopus egg extracts led to formation of colonies after bacteria transformation. As a control, mixing the linearized plasmid with egg extracts without incubation did not yield colony formation. ( C ) As in panel A, the linearized plasmid DNA was incubated in Xenopus egg extracts for 0.5 or 2 hr, re-isolated, and transformed into bacteria cells. The repair activity was measured by colony numbers. ( D ) The repair assay was performed in Xenopus egg extract that was supplemented with or without Ku70 antibody. The repair activity (in relative to the control extract) was measured by colony numbers. ( E ) The repair assay was performed in Xenopus egg extract that was supplemented with or without DNA-PKcs inhibitor (NU7441). The repair activity was measured by colony formation and compared between extracts with or without the inhibitor. Five types of repair templates were used, as in Fig. S2 . In panels C–E, a minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Journal: Scientific Reports

    Article Title: Non-homologous end joining repair in Xenopus egg extract

    doi: 10.1038/srep27797

    Figure Lengend Snippet: Repair of DSB substrates in Xenopus egg extract. ( A ) Schematic diagram of the DSB repair assay. As described in Materials and Methods, linearized plasmid DNA was generated and incubated in Xenopus egg extract. Plasmid DNA was then re-isolated from egg extracts, and transformed into bacteria cells. The final repair products were isolated from bacteria colonies and subjected to sequencing analysis. ( B ) As in panel A, incubation of plasmid DNA linearized with Xho1 and Kpn1 in Xenopus egg extracts led to formation of colonies after bacteria transformation. As a control, mixing the linearized plasmid with egg extracts without incubation did not yield colony formation. ( C ) As in panel A, the linearized plasmid DNA was incubated in Xenopus egg extracts for 0.5 or 2 hr, re-isolated, and transformed into bacteria cells. The repair activity was measured by colony numbers. ( D ) The repair assay was performed in Xenopus egg extract that was supplemented with or without Ku70 antibody. The repair activity (in relative to the control extract) was measured by colony numbers. ( E ) The repair assay was performed in Xenopus egg extract that was supplemented with or without DNA-PKcs inhibitor (NU7441). The repair activity was measured by colony formation and compared between extracts with or without the inhibitor. Five types of repair templates were used, as in Fig. S2 . In panels C–E, a minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Article Snippet: DNA repair assay in Xenopus egg extracts Plasmid pMBP-parallel2 (pMBPII) was digested with different restriction enzymes (New England Biolabs, MA) to generate linearized DNA with various end structures.

    Techniques: Plasmid Preparation, Generated, Incubation, Isolation, Transformation Assay, Sequencing, Activity Assay