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Determination of Mutation Frequency During Viral DNA Replication
病毒DNA复制过程中的突变频率测定   

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参见作者原研究论文

本实验方案简略版
Journal of Virology
Sep 2013

Abstract

This protocol is a simple method for evaluating mutation frequency during African swine fever virus (ASFV) replication, although it could be used also for other DNA viruses (poxvirus, herpesvirus, mimivirus, etc) with minor modifications. In the original Carrascosa et al. (1982), the protocol was carried out with two cloned viruses, BA71Vc (a purified clone from BA71V wild type strain) and vΔpolX (lacking the reparative polymerase, pol X, gene), and two different cell types that can be infected by ASFV, Vero cells and swine macrophages. To facilitate the sequence comparison, a genome fragment containing the B646L gene was amplified by PCR and blunt-end cloned. This gene codes for the major capsid protein (p72) and multiple sequences can be found in the database, so the mutations found could be compared with natural gene variations. The cloned fragment can be either sequenced directly from bacteria colonies or from miniprep purified DNA.

Keywords: DNA replication (DNA的复制), DNA repair (DNA修复), Fidelity (保真度), Oxidative DNA damage (DNA氧化损伤), Nucleo-citoplasmatic large DNA virus (核citoplasmatic大DNA病毒)

Materials and Reagents

  1. Virus supernatants (previously titrated)
  2. XL-1 Blue competent bacterial cells (Stratagene, catalog Number: 200249 )
  3. Dulbecco modified Eagle medium (DMEM) (Sigma-Aldrich, catalog number: D5523 )
  4. Fetal Bovine Serum (Sigma-Aldrich, catalog number: F7524 )
  5. Vero Cells (ATCC, catalog number: CCL-81 TM)
  6. Alveolar swine macrophages (Carrascosa et al., 1982)
  7. Swine serum (obtained during the macrophages purification)
  8. Kapa HiFi polymerase (Kapa Biosystems, catalog number: KK2101 )
    Note: This polymerase kit includes buffer and dNTPs.
  9. Primer B646L forward (Sigma-Aldrich)
    5’ATGGCATCAGGAGGAGCTTTTTGTCTTATT
  10. Primer B646L reverse (Sigma-Aldrich)
    5’TTAGGTACTGTAACGCAGCACAGCTGAACCG
  11. CloneJet PCR Cloning Kit (Thermo Fisher Scientific, catalog number: K1231 )
    Note: This kit includes the pJET plasmid, DNA ligase and reaction buffer.
  12. Agarose for DNA electrophoresis (Lonza, SeaKem® LE Agarose, catalog number: 50000 )
  13. TAE buffer (Sambrook and Russell, 2001)
  14. TE buffer (pH 8.0) (Sambrook and Russell, 2001)
  15. 5x Agarose Gel loading dye (QIAGEN, catalog number: 239901 )
  16. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
  17. LB-agar plates (Sambrook and Russell, 2001)
  18. Ampicillin (Sigma-Aldrich, catalog number: A0166 )
  19. Double-distillated or Milli-Q water

Equipment

  1. MW24 plates (BD Biosciences, Falcon®, catalog number: 353047 )
  2. 1.5 ml Eppendorf tubes
  3. Cell culture incubator
  4. Refrigerated benchtop centrifuge (e.g. Hettich 200 R)
  5. Thermal-cycler for PCR
  6. Electrophoresis system for agarose gels and UV-documentation device to visualize the DNA bands
  7. Waterbath

Software

  1. Diverse bioinformatics software (e.g., Geneious or CLC Genomics Workbench) (optional)
  2. MS Excel, Prism Graph Pad or SPSS (optional)

Procedure

  1. Sample preparations
    1. Plate Vero cells at about 30,000 cells/cm2 and swine macrophages at 60,000 cells/cm2 in multi-well 24 plate in DMEM-10% fetal-bovine or swine sera, respectively. Leave overnight in a cell incubator at 37 °C with 7.5% CO2.
      Note: For more details about ASFV infection protocols see Reference 4.
    2. Next day infect cultures at an moi = 5 pfu/cell.
    3. Prepare a negative control of uninfected cells and an extra well to monitor the infection success by titration. In the case of ASFV, we titrate the virus as in Reference 4. Other approaches could be used to evaluate the virus replication, such as qPCR.
    4. At 20 h post infection, when total cytopathic effect is achieved, harvest the cells and media in 1.5 ml Eppendorf tubes.
    5. Centrifuge 30 min at 14,000 rpm (about 20,000 x g) in Hettich (4 °C).
    6. Wash the pellets with ice-cold TE (about 200 μl) and spin again for 5 min.
    7. Suspend the pellet in 50 μl double-distilled water and boil for 20 min in a waterbath at 95 °C.
    8. Spin briefly and use supernatant as sample.
    9. Prepare the PCR tubes
      Component
      Per reaction (μl)
      5x Buffer
      5
      10 mM dNTPs
      0.75
      5 μM Primer forward
      5
      5 μM Primer reverse
      5
      Kapa HiFi DNA polymerase
      0.25
      Sample
      0.5
      Water
      8.5

  2. Set up the PCR program
    1. Adjust the annealing temperature according to the primer pair used. Increasing melting temperature may be required to avoid unspecific amplification products. Additionally, if there is no or very low amounts of amplified product an extra PCR step with 2-5 cycles at lower melting temperature may improve the results.
    2. Denaturation temperature and incubation time were optimized following the Kapa HiFi polymerase recommendations.
      Step 1

      1 cycle
      5 min
      95 °C

      Step 2 (optional)

      3 cycles
      1 min
      94 °C

      1 min
      47 °C

      5 min
      72 °C

      Step 3

      20 cycles
      30 sec
      98 °C

      30 sec
      54 °C

      4 min
      72 °C

      Step 4

      1 cycle
      15 min
      72 °C


    3. Run a small aliquot (2 μl) in agarose DNA electrophoresis to verify and quantify the amplification product.
    4. Run the PCR product in 0.7% high quality agarose, running the gel in 1x TAE buffer. Purify the PCR product by single band gel extraction using the QIAquick gel extraction kit (this step could be avoided, but the cloning efficiency might decrease).

  3. Ligation
    Use about 100 ng of PCR product and 0.5-1 μl pJET plasmid. The CloneJet kit includes 2x reaction buffer and T4 DNA ligase, although any ligase may be used. Manufacturer’s instructions recommended 5-30 min incubation at RT, but longer reactions (up to 2 h) may be done to increase the ligation yield.

  4. Bacteria transformation
    1. Transform XL-1 cells with the ligated plasmids and plate in LB-Agar plates with 100 μg/ml ampicillin.
    2. After 12-16 h incubation at 37 °C, pick colonies and suspend them in 5 μl TE for direct sequencing [performed as in Sambrook and Russell (2001)]. Take at least 15 colonies of each sample.
      Optional: Although ideally the cloning efficiency is about 80%, false positives for PCR fragments not purified from single band can be produced. In those cases, it is highly recommendable to inoculate 5-10 ml LB medium for miniprep purification of plasmids and to check the presence of the insert by restriction digestion (the plasmid contains BglII sites surrounding the ligation site).

  5. Data analysis
    Sequences can be checked by hand or assembled with diverse bioinformatics software. We recommend Geneious or CLC Genomics Workbench, which allow simultaneous display of the alignments and all sequencing peaks in order to differentiate the “mixed peaks” from true base changes.
    Example [see Table 1 in Carrascosa et al. (1982)]: We found 3 TA-CG transitions out of 56202 sequenced nucleotides, which give rise to a frequency of mutation of 5.34 x 10-5.
    The number of mutations is usually low and statistic analysis should be made with non-parametric test, like Mann-Whitney U, which is implemented in many different software packages, including MS Excel, Prism Graph Pad or SPSS.

Acknowledgments

This protocol is based on the procedure described in Redrejo-Rodríguez et al. (2013). This work was supported by a grant from the Spanish Ministerio de Economía y Competitividad (AGL2010-22229-C03-02) and by an institutional grant from Fundación Ramón Areces.

References

  1. Carrascosa, A. L., Santaren, J. F. and Vinuela, E. (1982). Production and titration of African swine fever virus in porcine alveolar macrophages. J Virol Methods 3(6): 303-310.
  2. de Leon, P., Bustos, M. J. and Carrascosa, A. L. (2013). Laboratory methods to study African swine fever virus. Virus Res 173(1): 168-179.
  3. Nelson, J. R., Cai, Y. C., Giesler, T. L., Farchaus, J. W., Sundaram, S. T., Ortiz-Rivera, M., Hosta, L. P., Hewitt, P. L., Mamone, J. A., Palaniappan, C. and Fuller, C. W. (2002). TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing. Biotechniques Suppl: 44-47.
  4. Redrejo-Rodriguez, M., Rodriguez, J. M., Suarez, C., Salas, J. and Salas, M. L. (2013). Involvement of the reparative DNA polymerase Pol X Of African swine fever virus in the maintenance of viral genome stability in vivo. J Virol 87(17): 9780-9787.
  5. Sambrook, J. and Russell, D. (2001). Molecular cloning: a laboratory manual, 4th ed. Cold Spring Harbor Laboratory Press. 

简介

该协议是用于评估非洲猪瘟病毒(ASFV)复制期间的突变频率的简单方法,尽管其也可以用于其它具有微小修改的DNA病毒(痘病毒,疱疹病毒,mimivirus,等)。 在原始Carrascosa等(1982)中,使用两种克隆的病毒BA71Vc(来自BA71V野生型菌株的纯化克隆)和vΔpolX(缺乏修复聚合酶,pol X, 基因)和可被ASFV,Vero细胞和猪巨噬细胞感染的两种不同细胞类型。 为了便于序列比较,通过PCR扩增含有B646L基因的基因组片段,并平端克隆。 该基因编码主要衣壳蛋白(p72),并且可以在数据库中发现多个序列,因此可以将发现的突变与天然基因变异进行比较。 克隆的片段可以直接从细菌菌落或从小量制备纯化的DNA测序。

关键字:DNA的复制, DNA修复, 保真度, DNA氧化损伤, 核citoplasmatic大DNA病毒

材料和试剂

  1. 病毒上清液(以前滴定)
  2. XL-1蓝色感受态细菌细胞(Stratagene,目录号:200249)
  3. Dulbecco改良的Eagle培养基(DMEM)(Sigma-Aldrich,目录号:D5523)
  4. 胎牛血清(Sigma-Aldrich,目录号:F7524)
  5. Vero细胞(ATCC,目录号:CCL-81 TM
  6. 肺泡猪巨噬细胞(Carrascosa et al。,1982)
  7. 猪血清(在巨噬细胞纯化期间获得)
  8. Kapa HiFi聚合酶(Kapa Biosystems,目录号:KK2101) 注意:此聚合酶试剂盒包括缓冲液和dNTPs。
  9. 引物B646L正向(Sigma-Aldrich)
    5'ATGGCATCAGGAGGAGCTTTTTGTCTTATT
  10. 引物B646L反向(Sigma-Aldrich)
    5'TTAGGTACTGTAACGCAGCACAGCTGAACCG
  11. CloneJet PCR克隆试剂盒(Thermo Fisher Scientific,目录号:K1231) 注意:该试剂盒包括pJET质粒,DNA连接酶和反应缓冲液。
  12. 用于DNA电泳的琼脂糖(Lonza,SeaKem LE agarose,目录号:50000)
  13. TAE缓冲液(Sambrook和Russell,2001)
  14. TE缓冲液(pH8.0)(Sambrook和Russell,2001)
  15. 5×琼脂糖凝胶负载染料(QIAGEN,目录号:239901)
  16. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  17. LB-琼脂平板(Sambrook和Russell,2001)
  18. 氨苄青霉素(Sigma-Aldrich,目录号:A0166)
  19. 双蒸馏水或Milli-Q水

设备

  1. MW24平板(BD Biosciences,Falcon ,目录号:353047)
  2. 1.5 ml Eppendorf管
  3. 细胞培养孵化器
  4. 制冷台式离心机(例如 Hettich 200 R)
  5. PCR热循环仪
  6. 琼脂糖凝胶电泳系统和UV记录装置可视化DNA条带
  7. 水浴

软件

  1. 不同的生物信息学软件(例如,,Geneious或CLC Genomics Workbench)(可选)
  2. MS Excel,Prism Graph Pad或SPSS(可选)

程序

  1. 样品制备
    1. 在多孔24孔板中在DMEM-10%胎牛或猪血清中培养约30,000细胞/cm 2的板Vero细胞和60,000细胞/cm 2的猪巨噬细胞 , 分别。 在37℃,7.5%CO 2的细胞培养箱中放置过夜 注意:有关ASFV感染协议的详细信息,请参阅参考文献4。
    2. 第二天以moi = 5pfu /细胞感染培养物
    3. 准备阴性对照的未感染细胞和额外的井,以监测通过滴定感染成功。 在ASFV的情况下,我们像参考文献4中那样滴定病毒。可以使用其他方法来评估病毒复制,例如qPCR。
    4. 在感染后20小时,当达到总细胞病变效应时,在1.5ml Eppendorf管中收获细胞和培养基。
    5. 在Hettich(4℃)中以14,000rpm(约20,000×g)离心30分钟。
    6. 用冰冷的TE(约200μl)洗涤沉淀,再次旋转5分钟
    7. 将沉淀悬浮在50μl双蒸水中,在95℃的水浴中煮沸20分钟。
    8. 简单旋转,并使用上清液作为样品
    9. 准备PCR管
      组件
      每反应(μl)
      5x缓冲区
      5
      10 mM dNTPs
      0.75
      5μM引物正向
      5
      5μM引物反转
      5
      Kapa HiFi DNA聚合酶
      0.25
      样品
      0.5

      8.5

  2. 设置PCR程序
    1. 根据使用的引物对调整退火温度。 可能需要增加熔化温度以避免非特异性扩增产物。 此外,如果没有或非常少量的扩增产物,在较低熔解温度下用2-5个循环的额外PCR步骤可以改善结果。
    2. 变性温度和温育时间根据Kapa HiFi聚合酶推荐进行优化。
      步骤1

      1个周期
      5分钟
      95°C

      步骤2(可选)

      3个周期
      1分钟
      94°C

      1分钟
      47℃

      5分钟
      72℃

      步骤3

      20个周期
      30秒
      98°C

      30秒
      54°C

      4分钟
      72℃

      步骤4

      1个周期
      15分钟
      72℃


    3. 在琼脂糖DNA电泳中运行一小份等分试样(2μl)以验证和定量扩增产物
    4. 在0.7%高品质琼脂糖中运行PCR产物,在1x TAE缓冲液中运行凝胶。 使用QIAquick凝胶提取试剂盒通过单带凝胶提取纯化PCR产物(此步骤可以避免,但克隆效率可能会降低)。

  3. 连接
    使用约100ng的PCR产物和0.5-1μlpJET质粒。 CloneJet试剂盒包括2x反应缓冲液和T4DNA连接酶,尽管可以使用任何连接酶。 制造商的说明书建议在室温下孵育5-30分钟,但可以进行更长的反应(长达2小时)以增加连接产量。
  4. 细菌转化
    1. 用连接的质粒转化XL-1细胞,并在具有100μg/ml氨苄青霉素的LB-琼脂平板中平板。
    2. 在37℃下孵育12-16小时后,挑选菌落并将其悬浮于5μlTE中以进行直接测序[如Sambrook和Russell(2001)中所述进行]。取每个样品至少15个菌落。
      可选:虽然理想情况下克隆效率为约80%,但是可以产生不从单条带纯化的PCR片段的假阳性。在这些情况下,强烈推荐接种5-10ml LB培养基以用于质粒的小量制备纯化,并通过限制性消化(质粒含有连接位点周围的BglII位点)检查插入片段的存在。

  5. 数据分析
    可以手工检查或用不同的生物信息学软件装配序列。我们建议Geneious或CLC Genomics Workbench,它们允许同时显示比对和所有测序峰,以区分"混合峰"与真正的碱基变化。
    实施例[参见Carrascosa et al。 (1982)]:我们在56202个测序核苷酸中发现了3个TA-CG转换,其导致突变为5.34×10 -5 s -1 的频率。 突变的数量通常较低,并且应该用非参数检验进行统计分析,如Mann-Whitney U,其在许多不同的软件包中实现,包括MS Excel,Prism Graph Pad或SPSS。

致谢

该协议基于Redrejo-Rodríguez等人(2013)中描述的程序。这项工作得到了西班牙经济部长竞争力奖(AGL2010-22229-C03-02)和拉美区域基金会的资助。

参考文献

  1. Carrascosa,A.L.,Santaren,J.F.and Vinuela,E。(1982)。 在猪肺泡巨噬细胞中产生和滴定非洲猪瘟病毒。病毒学方法 3(6):303-310
  2. de Leon,P.,Bustos,M.J。和Carrascosa,A.L。(2013)。 研究非洲猪瘟病毒的实验室方法 病毒研究 173(1):168-179。
  3. Nelson,JR,Cai,YC,Giesler,TL,Farchaus,JW,Sundaram,ST,Ortiz-Rivera,M.,Hosta,LP,Hewitt,PL,Mamone,JA,Palaniappan,C.and Fuller, 。 TempliPhi,phi29基于DNA聚合酶的滚环扩增用于DNA测序的模板。 Biotechniques Suppl :44-47
  4. Redrejo-Rodriguez,M.,Rodriguez,J. M.,Suarez,C.,Salas,J.and Salas,M.L。(2013)。 涉及非洲猪瘟病毒的修复性DNA聚合酶Pol X维持病毒基因组稳定性< em> J Virol 87(17):9780-9787。
  5. Sambrook,J。和Russell,D。(2001)。 分子克隆:实验室手册,4版 冷泉港实验室出版社。
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引用:Redrejo-Rodríguez, M., Rodríguez, J. M., Suárez, C. and Salas, M. L. (2014). Determination of Mutation Frequency During Viral DNA Replication. Bio-protocol 4(6): e1076. DOI: 10.21769/BioProtoc.1076.
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