DNA 损伤类型和原因 |NEB酶试剂 New England Biolabs

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产品信息

PreCR® 修复混合液是一种酶混合试剂，用于在聚合酶链式反应（PCR）、微阵列分析或其它 DNA 技术之前，修复受损的 DNA 模板。PreCR 作用于多种受损 DNA，包括那些阻断 PCR 反应的损伤（如：脱嘌呤/脱嘧啶位点、胸腺嘧啶二聚体、切刻和缺口）和致突变的 DNA 损伤（如脱氨基胞嘧啶和 8-氧鸟嘌呤）。此外，它能去除 DNA 3´ 末端的多种基团而留下羟基基团。PreCR 修复混合液不能修复所有抑制和干扰 PCR 的损伤。例如，其不会修复 8-氧-7,8-二羟基-2´-脱氧腺苷或片段化 DNA。事实上，混合液中的连接酶能有效连接 DNA 切刻，但不能有效连接平末端或错配附近的切刻。这限制了连接产物形成嵌合基因的可能性。PreCR 修复混合液可以与任何一种嗜热聚合酶配合使用。

暴露于各种化学或环境因素、操作或自然老化，让 DNA 容易遭受多种类型的损伤。DNA 损伤和 PreCR 表列出了一些可能的 DNA 样本及其可能遭受的损伤的影响。

注意：暴露于不同试剂所造成的损伤程度可能不同，其重要性将取决于 DNA 的使用方式。

表 1： DNA 损伤类型和原因 |
DNA 损伤类型
图 1：用 PreCR 修复混合液修复不同类型的 DNA 损伤。 DNA 损伤类型和原因 |
胶图显示了未经过 PreCR 修复混合液处理（-）或已经过 PreCR 修复混合液处理（+）的受损 DNA 的扩增结果。DNA 损伤的类型显示在图上方。注意：热处理 DNA 为 99℃ 温育 3 分钟。Marker (M) 为 1 kb Plus DNA Ladder（NEB #N3200）。

产品类别:
DNA Manipulation Products,
DNA Repair Enzymes and Structure-specific Endonucleases Products

应用:
PCR,
PCR

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • M0309S     -20    
        PreCR® Repair Mix M0309SVIAL -20 1 x 0.03 ml 30 reactions
        β-Nicotinamide adenine dinucleotide (NAD+) B9007SVIAL -20 1 x 0.2 ml 100 X
        ThermoPol® Reaction Buffer B9004SVIAL -20 1 x 1.5 ml 10 X
        UV damaged Lambda DNA  N3017AVIAL -20 1 x 0.06 ml 0.5 µg/ml
        L1 Primer Mix S1284AVIAL -20 1 x 0.02 ml 20 µM
    • M0309L     -20    
        PreCR® Repair Mix M0309LVIAL -20 1 x 0.15 ml 150 reactions
        β-Nicotinamide adenine dinucleotide (NAD+) B9007SVIAL -20 1 x 0.2 ml 100 X
        ThermoPol® Reaction Buffer B9004SVIAL -20 1 x 1.5 ml 10 X
        UV damaged Lambda DNA  N3017AVIAL -20 1 x 0.06 ml 0.5 µg/ml
        L1 Primer Mix S1284AVIAL -20 1 x 0.02 ml 20 µM

  • 优势和特性

    应用特性

    • 在使用 DNA 作为 PCR 模板或其它 DNA 技术之前修复该 DNA。

  • 相关产品

    相关产品

    • Taq 2X 预混液
    • Taq DNA 连接酶
    • m0267-taq-dna-polymerase-with-thermopol-buffer
    • TriDye™ 1 kb Plus DNA Ladder
    • 核酸内切酶 IV
    • 核酸内切酶 VIII
    • 8-氧代鸟嘌呤 DNA 糖基酶（Fpg）
    • Quick-Load® 1 kb Plus DNA Ladder
    • T4 PDG 嘧啶二聚体糖基酶（T4 核酸内切酶 V）
    • 尿嘧啶-DNA 糖基酶（UDG）（也称：尿嘧啶-DNA 糖基化酶）
    • m0328-bst-dna-polymerase-full-length
    • dNTP 混合液

    单独销售的组分

    • β-烟酰胺腺嘌呤二核苷酸（NAD+）
    • ThermoPol® 反应缓冲液套装

  • 注意事项

    1. * PreCR 修复混合液成分：Taq DNA 连接酶、核酸内切酶 IV、Bst DNA 聚合酶、8-氧代鸟嘌呤 DNA 糖基化酶（Fpg）、尿嘧啶-DNA 糖基化酶（UDG）、T4 PDG 嘧啶二聚体糖基化酶（T4 核酸内切酶 V）和核酸内切酶 VIII。
    2. 反应条件：DNA 模板、100 µM dNTP、1X ThermoPol 反应缓冲液、1X NAD+ 和 1 µl PreCR 修复混合液，总反应体积为 50 µl。37℃ 温育。
    3. 1X NAD+ 溶液：0.5 mM NAD+。
    4. PreCR 修复混合液中不包含 dNTP。

  • 参考文献

    1. Diegoli TM, Farr M, Cromartie C, Coble MD, Bille TW. (2012). An optimized protocol for forensic application of the PreCR™ Repair Mix to multiplex STR amplification of UV-damaged DNA.. Forensic Sci Int Genet. Jul, 498-503. PubMedID: 22001155

操作说明、说明书 & 用法

  • 操作说明

    1. Sequential Reaction Protocol for PreCR Repair Mix
    2. Standard Reaction Protocol for PreCR Repair Mix
    3. Control Reaction Protocol for PreCR Repair Mix

  • 使用指南

    • DNA Damage and PreCR

FAQs & 问题解决指南

  • FAQs

    1. Will PreCR ligate my DNA fragments?
    2. How much DNA will PreCR repair?
    3. Will treating my DNA with the PreCR Repair Mix hurt my reaction?
    4. What is the sequence of the L1 primer mix?
    5. How is the damaged DNA that you test the PreCR Repair Mix against created? Can I buy this damaged DNA separately?
    6. Why does my control reaction not give a detectable amplicon?
    7. Can I buy any of the PreCR Repair Mix components separately?
    8. Will the PreCR Repair Mix blunt the ends of the DNA?
    9. Why don’t I see the expected band from my repaired template?
    10. Is my buffer compatible and which reaction should I use?
    11. Does the PreCr Repair Mix insert random nucleotides into the sequence that it repairs?
    12. Does the PreCR Repair Mix contain any contaminating human DNA? What are the quality controls that are used to test that?
    13. Does the PreCR Repair Mix remove covalent modifications from DNA bases, such as biotin or digoxigenin? Does it repair mismatches/ extra bases in DNA?
    14. If there are gaps and nicks remaining from a ligation reaction, will the PreCR Repair Mix repair all of these, so that the DNA will be suitable for microinjection into mice, for example?
    15. Can the PreCR Repair Mix be used for paraffin-embedded DNA?
    16. The repaired DNA will be used for an Nsp1 or Sty1 digestion followed by an adapter ligation, and PCR. Do you recommend cleanup of the PreCR Repair Mix reaction prior to this process?
    17. Can the PreCR Repair Mix repair damage in both single and double stranded DNA? Or, does it require a double stranded DNA as a template?
    18. Is the addition of dNTPs necessary for the PreCR Repair Mix to work properly?
    19. If I had a DNA template with mutation sites (ie. 8-oxoguanine or deaminated cytosines) that are directly adjacent to each other on opposite strands would treatment with PreCR™ Repair Mix cause a double strand nick/break?
    20. What gap lengths can be repaired with the PreCR Repair Mix?
    21. Does the PreCR Repair Mix work with less concentrated amounts of DNA (e.g. 500pg-1ng) than the amounts recommended?
    22. When doing bisulfite treatments of templates, the subsequent PCR reactions can pose a problem as the DNA seems to be very labile after the treatment. Can the PreCR Repair Mix improve these PCR results?
    23. Is it necessary to clean the PreCR reaction prior to carrying out quantitative PCR?
    24. When working with fragments, will the ends be ligated together by the PreCR Reaction Mix?
    25. Can T4 DNA ligase be used to ligate across an abasic site?
    26. How are abasic sites repaired by the PreCR Reaction Mix? Are new nucleotides put in and ligated? Is the existing nucleotide repaired?

  • 问题解决指南

    • PCR Troubleshooting Guide