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PCR-based Assay for Genome Integrity after Methyl Methanesulfonate Damage in Physcomitrella patens
PCR检测研究小立碗藓遭受总甲磺酸甲酯损坏后的基因组完整性   

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Abstract

In plant cells, genomic DNA exists in three organelles: the nucleus, chloroplast, and mitochondrion. Genomic DNA can be damaged by endogenous and exogenous factors, but the damaged DNA can be repaired by DNA repair systems. To quantify the extent of their repair activity of on individual genomic DNA, a PCR-based assay utilizing long amplicons is valuable for evaluable. This assay is based on the inhibitory effects of methyl methanesulfonate (MMS)-induced DNA damage on the amplicons. This assay is useful for assessing DNA double-strand repair pathways, such as homologous recombination repair, as it detects DNA double-strand breaks produced by MMS in vivo.

Keywords: Chloroplast(叶绿体), Genome stability(基因组稳定性), PCR(PCR), DNA damage(DNA损伤), Physcomitrella patens(小立碗藓)

Background

The quantification of genomic DNA damage is useful for analyzing DNA repair mechanisms. This assay utilizes real-time PCR to quantify the nuclear, chloroplast, and mitochondrial DNA copy number for the normalization of long PCR products, providing more accurate quantification compared with that by the previous protocol by Hunter et al. (2010).

Materials and Reagents

  1. 90 mm plastic Petri dish
  2. 50 ml tube
  3. 1.5 ml tube
  4. Physcomitrella patens protonemal cells (cultivated for 4 days)
  5. BCDAT medium (Nishiyama et al., 2000)
  6. Methyl methanesulfonate (MMS) (Sigma-Aldrich, catalog number: 129925 )
  7. Chloroform (Sigma-Aldrich, catalog number: V800117 )
  8. Ethanol (Sigma-Aldrich, catalog number: 09-0770 )
  9. RNase A
  10. LA Taq (TAKARA BIO, catalog number: RR002A )
  11. Agarose powder (Promega, catalog number: V3125 )
  12. 0.7% agarose gel
  13. Ethidium bromide (EtBr)
  14. Power SYBR® green PCR master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4367659 )
  15. Hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H-5882 )
  16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 28-2270 )
  17. Tris-HCl, pH 8.0 (Wako Pure Chemical Industries, catalog number: 206-07884 )
  18. Ethylenediamine-N,N,N’,N’-tetraacetic acid, disodium salt, dihydrate (EDTA) (DOJINDO, catalog number: 345-01865 )
  19. PEG8000 (MP Biomedicals, catalog number: 0 2194839 )
  20. Oligonucleotide DNA primers for long amplicon quantitative PCR:
    Nuclear DNA (5′-GTGGAAGTGAGATGCAGTTTGG-3′ and 5'-GTGGCTCTGGACAGTGAAATTG-3′)
    Chloroplast DNA (5′-AATTGGAGTCGGTCCTTCCATA-3′ and 5′-TGGCAAATACAATGGCAAAAAG-3′)
    Mitochondrial DNA (5′-GACTGCCCTCACTAGGATGCTT-3′ and 5′-TGGGTGATTTACTCCATTGACG-3′)
  21. Oligonucleotide DNA primers for short amplicon quantitative PCR:
    Nuclear DNA (5′-CAACCGTCTTCTGTGTCTAGGTC-3′ and 5′-GAAACCGGCCTGCATTACATG-3′)
    Chloroplast DNA (5′-AGTAATCCCATCGCGTGACAT-3′ and 5′-AGGTATGGAAAAAATCGCTGAAAA-3′)
    Mitochondrial DNA (5′-CGTGCTAAAAATCCAGTCCATTC-3′ and 5′-AGCAAAGAAGTCAAGACCTAACAAAAC-3′)
  22. 2x CTAB buffer (see Recipes)
  23. 10% CTAB (see Recipes)
  24. CTAB ppt buffer (see Recipes)
  25. NaCl-TE (see Recipes)
  26. TE buffer (see Recipes)
  27. PEG solution (see Recipes)

Equipment

  1. Forceps
  2. Multi-beads shocker (Yasui Kikai)
  3. Vacuum pump (Tokyo Rikakikai, catalog number: AS-3 )
  4. Bench top centrifuge for 1.5 ml tubes
  5. Heat block for 1.5 ml tubes (TAITEC, catalog number: DTU-2CN )
  6. Standard thermal cycler (Bio-Rad Laboratories, catalog number: 170-8720JA )
  7. Real-time PCR thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: Applied Biosystems® 7500 Fast Real-Time PCR Systems)

Software

  1. ImageJ (https://imagej.nih.gov/ij/)
  2. Excel (Microsoft)

Procedure

  1. MMS treatment of plants
    1. Cultivate P. patens protonemal cells on four BCDAT agar plates for each strain under continuous light at 25 °C.
    2. Immerse P. patens protonemal cells in 12 ml BCDAT liquid medium containing 20 mM MMS in 90 mm plastic Petri dishes under vacuum for 1 h. Note that undamaged control samples are harvested prior to the MMS treatment. These should include three biological replicates.
      Note: MMS is a carcinogen.
    3. After washing with BCDAT liquid medium (twice with 15 ml BCDAT liquid medium in 50 ml tubes), cultivating the protonemal cells under continuous light at 25 °C.
    4. Using forceps, harvest the cells at 0, 6, 24 and 48 h after washing.

  2. Extraction of total genomic DNA
    1. Grind frozen protonemal cells using a homogenizer (Multi-beads shocker: 1,500 rpm, 10 sec, thrice). Alternatively, mortar and pestle can be used.
    2. Add an equal volume (approximately 300 µl in a 1.5 ml tube) of 2x CTAB buffer to the powdered protonemal cells. Mix and then incubate at 60 °C for 1 h.
    3. Mix with an equal volume of chloroform and centrifuge at 4,500 x g for 20 min. Recover aqueous phase (approximately 500 µl).
    4. Add 1/10 volume of 10% CTAB.
    5. Extract DNA with an equal volume of chloroform and recover the aqueous phase. Repeat this step twice.
    6. Add an equal volume of CTAB ppt buffer and mix well by inverting.
    7. Centrifuge at 4,500 x g for 20 min. Discard the supernatant.
    8. Dissolve the pellet in 400 µl of NaCl-TE. Incubate at 60 °C for 30 min.
    9. Add two volumes of ethanol.
    10. Centrifuge at 14,000 x g for 10 min. Discard the supernatant.
    11. Dissolve the pellet in 100 µl of TE. The pellet may be insoluble at this step, but will dissolve in the next step.
    12. Add 2 µl of RNase A (1 mg/ml) and incubate at 37 °C for 1 h.
    13. Add an equal volume of PEG solution.
    14. Centrifuge at 14,000 x g at 4 °C for 10 min.
    15. After removing the supernatant, rinse the pellet with 70% ethanol.
    16. Dissolve the pellet in 100 µl of TE.

  3. Long amplicon quantitative PCR
    Long amplicon PCR is performed to quantify the amount of undamaged DNA template.
    1. Design primers to generate 8-10 kb PCR amplicons from the nuclear, chloroplast, and mitochondrial genomes.
      Note: Long amplicons efficiently detect DNA damage; however, amplification efficiency is poor.
    2. Prepare a dilution series (20, 10, 5, 2.5, 1.25 and 0.625 ng/µl by diluting with TE buffer) of undamaged DNA, spanning the range from the lowest to the highest amount of PCR product in the damaged samples, to use as standard. Diluted samples to 10 ng/µl by adding TE buffer, and use 1 µl for each PCR reaction.
    3. Perform a 2-step PCR (first step, 98 °C for 10 sec; second step, 68 °C for 10 min) using a Taq DNA polymerase optimized for long amplicon (LA-Taq). Subject all samples and control dilutions to a single PCR reaction. Use the number of PCR cycles at which the resulting DNA amplicon is exponentially amplified. Thus, the resulting amount/yield of the PCR amplicon reflects the amount of DNA template used in the reaction. Use 17, 20 and 27 cycles for amplifying the chloroplast, mitochondria and nuclear DNA, respectively.
    4. Electrophorese on 0.7% agarose gels stained with EtBr to resolve the PCR products (Figure 1). Load a suitable fraction of the PCR product on the gel.


      Figure 1. Electrophoresis of long PCR amplicons on agarose gel. An example of agarose gel electrophoresis of long PCR products from nuclear actin locus. No MMS damage control (no) and MMS-damaged samples (0, 6, 24 and 48 h after washing) were analyzed with serially diluted standard samples. RECA2-KO is shown as an example of a mutant (Odahara et al., 2015).

  4. Short amplicon quantitative PCR
    Short amplicon PCR allows copy number quantification of the nuclear, chloroplast and mitochondrial DNA. Both standard and real-time PCRs are applicable for this short amplicon quantitative PCR but the latter provides more accurate values.
    1. Design primers to amplify approximately 100 bp of the nuclear, chloroplast and mitochondrial loci, based on the standard protocol for quantitative real-time PCR assay using intercalating dyes, such as SYBR Green I.
    2. Prepare a dilution series to use as standard controls, including all sample concentrations. It is important to use the 10 ng/µl sample DNA prepared in the long amplicon PCR.
    3. Perform real-time PCR using DNA polymerase mixture optimized for real-time PCR (Power SYBR® Green PCR Master Mix) and thermal cycler real-time PCR system with a PCR program (first step, 95 °C for 15 sec; second step, 60 °C for 1 min). Calculate the relative copy number of the nuclear, chloroplast, and mitochondrial DNA of each sample using standard curves prepared using the standard samples.

Data analysis

Analysis of recovery from MMS damage
  1. Measure the volume of each band in the electrophoresis gel with ImageJ using the “measure” tool, and calculate the relative amount of long PCR products from all samples by fitting them to the standard curve prepared using those from the serially diluted standard samples.
  2. Divide the relative amount of long amplicon PCR products by the relative copy number of the nuclear, chloroplast and mitochondrial DNA calculated from short amplicon PCR products to obtain the relative copy number of undamaged DNA.
  3. Relative recovery is calculated by dividing the amount of undamaged DNA in damaged samples by that of undamaged controls. Use t-test for statistical analysis of data from three biological replicates and Excel.

Recipes

  1. 2x CTAB buffer
    2% CTAB
    1.4 M NaCl
    100 mM Tris-HCl, pH 8.0
    20 mM EDTA
    Store at room temperature (RT).
  2. 10% CTAB
    10% CTAB
    0.7 M NaCl
    Store at RT.
  3. CTAB ppt buffer
    1% CTAB
    50 mM Tris-HCl, pH 8.0
    10 mM EDTA
    Store at RT.
  4. NaCl-TE
    1 M NaCl
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA
    Store at RT.
  5. TE buffer
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA
    Store at RT.
  6. PEG solution
    2 M NaCl
    20% PEG8000
    Store at RT.

Acknowledgments

The long amplicon PCR assay is based on Hunter et al. (2010) with slight modifications. The genomic DNA extraction protocol is based on PHYSCObase (http://moss.nibb.ac.jp/). This work was supported by the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Funding Program for Next-Generation World-Leading Researchers (NEXT Program).

References

  1. Hunter, S. E., Jung, D., Di Giulio, R. T. and Meyer, J. N. (2010). The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number. Methods 51(4): 444-451.
  2. Nishiyama, T., Hiwatashi, Y., Sakakibara, I., Kato, M. and Hasebe, M. (2000). Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res 7(1): 9-17.
  3. Odahara, M., Inouye, T., Nishimura, Y. and Sekine, Y. (2015). RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens. Plant J 84(3): 516-526.

简介

在植物细胞中,基因组DNA存在于三个细胞器中:核,叶绿体和线粒体。基因组DNA可以被内源和外源因子损伤,但损伤的DNA可以通过DNA修复系统修复。为了量化其对单个基因组DNA的修复活性的程度,使用长扩增子的基于PCR的测定对于可评价是有价值的。该测定基于甲磺酸甲酯(MMS)诱导的DNA损伤对扩增子的抑制作用。该测定可用于评估DNA双链修复途径,例如同源重组修复,因为其检测由MMS在体内产生的DNA双链断裂。

[背景] 基因组DNA损伤的定量可用于分析DNA修复机制。该测定利用实时PCR定量核,叶绿体和线粒体DNA拷贝数以用于长PCR产物的标准化,与由Hunter等人先前的方案相比提供更准确的定量。 (2010)。

关键字:叶绿体, 基因组稳定性, PCR, DNA损伤, 小立碗藓

材料和试剂

  1. 90 mm塑料培养皿
  2. 50ml管
  3. 1.5毫升管
  4. 展叶剑叶藓质子细胞(培养4天)
  5. BCDAT培养基(Nishiyama等人,2000)
  6. 甲磺酸甲酯(MMS)(Sigma-Aldrich,目录号:129925)
  7. 氯仿(Sigma-Aldrich,目录号:V800117)
  8. 乙醇(Sigma-Aldrich,目录号:09-0770)
  9. RNase A
  10. LA Taq(TAKARA BIO,目录号:RR002A)
  11. 琼脂糖粉末(Promega,目录号:V3125)
  12. 0.7%琼脂糖凝胶
  13. 溴化乙锭(EtBr)
  14. Power SYBR 绿色PCR主混合物(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4367659)
  15. 十六烷基三甲基溴化铵(CTAB)(Sigma-Aldrich,目录号:H-5882)
  16. 氯化钠(NaCl)(Sigma-Aldrich,目录号:28-2270)
  17. Tris-HCl,pH8.0(Wako Pure Chemical Industries,目录号:206-07884)
  18. 乙二胺-N,N,N',N'-四乙酸二钠盐二水合物(EDTA)(DOJINDO,目录号:345-01865)
  19. PEG8000(MP Biomedicals,目录号:02194839)
  20. 用于长扩增子定量PCR的寡核苷酸DNA引物:
    核DNA(5'-GTGGAAGTGAGATGCAGTTTGG-3'和5'-GTGGCTCTGGACAGTGAAATTG-3')
    叶绿体DNA(5'-AATTGGAGTCGGTCCTTCCATA-3'和5'-TGGCAAATACAATGGCAAAAAG-3')
    线粒体DNA(5'-GACTGCCCTCACTAGGATGCTT-3'和5'-TGGGTGATTTACTCCATTGACG-3')
  21. 用于短扩增子定量PCR的寡核苷酸DNA引物:
    核DNA(5'-CAACCGTCTTCTGTGTCTAGGTC-3'和5'-GAAACCGGCCTGCATTACATG-3')
    叶绿体DNA(5'-AGTAATCCCATCGCGTGACAT-3'和5'-AGGTATGGAAAAAATCGCTGAAAA-3')
    线粒体DNA(5'-CGTGCTAAAAATCCAGTCCATTC-3'和5'-AGCAAAGAAGTCAAGACCTAACAAAAC-3')
  22. 2x CTAB缓冲液(参见配方)
  23. 10%CTAB(参见配方)
  24. CTAB ppt缓冲区(参见配方)
  25. NaCl-TE(参见配方)
  26. TE缓冲区(参见配方)
  27. PEG溶液(参见配方)

设备

  1. 镊子
  2. 多珠冲击器(Yasui Kikai)
  3. 真空泵(Tokyo Rikakikai,目录号:AS-3)
  4. 台式离心机用于1.5 ml管
  5. 用于1.5ml管(TAITEC,目录号:DTU-2CN)的加热块
  6. 标准热循环仪(Bio-Rad Laboratories,目录号:170-8720JA)
  7. 实时PCR热循环仪(Thermo Fisher Scientific,Applied Biosystems ,型号:Applied Biosystems 7500 Fast Real-Time PCR Systems)

软件

  1. ImageJ( https://imagej.nih.gov/ij/
  2. Excel(Microsoft)

程序

  1. MMS处理植物
    1. 培养 p。 patens 质粒细胞在四个BCDAT琼脂平板上,在25℃连续光照下。
    2. Immerse P。 patens质粒细胞在含有20mM MMS的12ml BCDAT液体培养基中在90mm塑料培养皿中真空培养1小时。注意,在MMS处理之前收获未受损的对照样品。这些应包括三个生物学重复 注意:MMS是致癌物质。
    3. 用BCDAT液体培养基(50ml试管中用15ml BCDAT液体培养基洗两次)洗涤后,在25℃连续光照下培养质子细胞。
    4. 使用镊子,在洗涤后0,6,24和48小时收获细胞
  2. 提取总基因组DNA
    1. 使用匀浆器(Multi-beads shocker:1,500rpm,10秒,三次)研磨冷冻的质子细胞。或者,可以使用研钵和杵
    2. 加入等体积(大约300微升在1.5毫升管)的2×CTAB缓冲液到粉末状的质子细胞。混合,然后在60℃下孵育1小时
    3. 与等体积的氯仿混合并在4,500×g离心20分钟。回收水相(约500μl)
    4. 加入1/10体积的10%CTAB
    5. 用等体积的氯仿提取DNA并回收水相。重复此步骤两次。
    6. 加入等体积的CTAB ppt缓冲液,倒转混匀
    7. 以4500xg离心20分钟。弃去上清液。
    8. 将沉淀溶解在400μlNaCl-TE中。在60℃孵育30分钟。
    9. 加入两倍体积的乙醇。
    10. 以14,000×g离心10分钟。弃去上清液。
    11. 将沉淀溶解在100μlTE中。颗粒在此步骤可能不溶,但在下一步骤中溶解
    12. 加入2微升的核糖核酸酶A(1毫克/毫升),并在37℃下孵育1小时
    13. 加入等体积的PEG溶液。
    14. 在4℃下以14,000×g离心10分钟。
    15. 除去上清液后,用70%乙醇冲洗沉淀
    16. 将沉淀溶于100μlTE中。

  3. 长扩增子定量PCR
    进行长扩增子PCR以定量未损伤的DNA模板的量
    1. 设计引物以从核,叶绿体和线粒体基因组产生8-10kb PCR扩增子。
      注意:长的扩增子有效地检测DNA损伤;然而,扩增效率很差。
    2. 准备一个稀释系列(20,10,5,2.5,1.25和0.625 ng /μl,通过用TE缓冲液稀释)未受损的DNA,跨越受损样品中从最低到最高量的PCR产物的范围,用作标准。通过加入TE缓冲液将样品稀释至10 ng /μl,每次PCR反应使用1μl
    3. 使用针对长扩增子(LA-Taq)优化的Taq DNA聚合酶进行2步PCR(第一步,98℃10秒;第二步,68℃10分钟)。将所有样品和对照稀释液进行单个PCR反应。使用所得DNA扩增子以指数方式扩增的PCR循环数。因此,PCR扩增子的所得量/产量反映了反应中使用的DNA模板的量。使用17,20和27个循环分别扩增叶绿体,线粒体和核DNA
    4. 在用EtBr染色的0.7%琼脂糖凝胶上电泳以分辨PCR产物(图1)。将适当部分的PCR产物装载在凝胶上

      图1.琼脂糖凝胶上的长PCR扩增子的电泳来自核肌动蛋白基因座的长PCR产物的琼脂糖凝胶电泳的实例。用连续稀释的标准样品分析没有MMS损伤对照(无)和MMS损伤的样品(洗涤后0,6,24和48小时)。 RECA2 -KO作为突变体的例子显示(Odahara等人。,2015)。

  4. 短扩增子定量PCR
    短扩增子PCR允许核,叶绿体和线粒体DNA的拷贝数量化。标准和实时PCR适用于这种短扩增子定量PCR,但后者提供更准确的值
    1. 基于使用插入染料(例如SYBR Green I)的定量实时PCR测定的标准方案,设计引物以扩增约100bp的核,叶绿体和线粒体基因座。
    2. 准备稀释系列用作标准对照,包括所有样品浓度。使用在长扩增子PCR中制备的10ng /μl样品DNA是重要的
    3. 使用针对实时PCR优化的DNA聚合酶混合物(Power SYBR Green PCR Master Mix)和热循环仪实时PCR系统,使用PCR程序进行实时PCR(第一步,95℃ 15秒;第二步,60℃1分钟)。使用标准样品制备的标准曲线计算每个样品的核,叶绿体和线粒体DNA的相对拷贝数。

数据分析

分析来自MMS损害的恢复
  1. 使用"测量"工具用ImageJ测量电泳凝胶中每条带的体积,并通过将它们与使用来自连续稀释的标准样品的那些拟合的标准曲线拟合,计算来自所有样品的长PCR产物的相对量。 />
  2. 将长扩增子PCR产物的相对量除以从短扩增PCR产物计算的核,叶绿体和线粒体DNA的相对拷贝数,以获得未损伤DNA的相对拷贝数。
  3. 通过将损伤样品中未损伤的DNA的量除以未损伤的对照的未损伤的DNA的量来计算相对恢复。使用 t -test对来自三个生物复制和Excel的数据进行统计分析

食谱

  1. 2x CTAB缓冲区
    2%CTAB
    1.4 M NaCl
    100 mM Tris-HCl,pH 8.0
    20 mM EDTA
    储存在室温(RT)。
  2. 10%CTAB
    10%CTAB
    0.7 M NaCl
    存储在RT。
  3. CTAB ppt缓冲区
    1%CTAB
    50 mM Tris-HCl,pH 8.0
    10 mM EDTA
    存储在RT。
  4. NaCl-TE
    1 M NaCl
    10mM Tris-HCl,pH8.0 1mM EDTA
    存储在RT。
  5. TE缓冲区
    10mM Tris-HCl,pH8.0 1mM EDTA
    存储在RT。
  6. PEG溶液
    2 M NaCl
    20%PEG8000
    存储在RT。

致谢

长扩增子PCR测定基于Hunter等人。 (2010)略有修改。基因组DNA提取方案基于PHYSCObase( http://moss.nibb.ac .jp/)。这项工作得到了日本教育,文化,体育,科学和技术部的战略研究基金赠款辅助项目以及下一代世界领先研究人员资助计划(NEXT计划)的支持。

参考文献

  1. Hunter,SE,Jung,D.,Di Giulio,RT和Meyer,JN(2010)。  用于分析线粒体DNA损伤,修复和相对拷贝数的QPCR测定法 51(4):444-451。 >
  2. Nishiyama,T.,Hiwatashi,Y.,Sakakibara,I.,Kato,M.and Hasebe,M。(2000)。  通过穿梭诱变在青苔,小立碗藓中标记诱变和基因陷阱。

    em> 7(1):9-17
  3. Odahara,M.,Inouye,T.,Nishimura,Y.和Sekine,Y.(2015)。  RECA在小立碗藓中维持叶绿体基因组稳定性中起双重作用。 ):516-526。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Odahara, M., Inouye, T., Nishimura, Y. and Sekine, Y. (2016). PCR-based Assay for Genome Integrity after Methyl Methanesulfonate Damage in Physcomitrella patens. Bio-protocol 6(19): e1954. DOI: 10.21769/BioProtoc.1954.
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