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In vitro DNA Polymerization Activity Assay Using Cell-free Extracts
采用细胞抽提液进行体外DNA聚合活性测试

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Abstract

This protocol has been designed to measure the in-vitro DNA polymerization activity in crude cell extracts of the Antarctic bacterium Pseudomonas syrinagae Lz4W. This bacterium can grow at 4 °C with optimum growth rate at 22 °C. The slow growth rate of the bacterium observed at low temperature (4 °C) compared to higher temperature (22 °C) can be attributed to the reduced rate of DNA replication at low temperature. Here we describe a protocol which we have used to quantify the in vitro DNA polymerization of cell extracts at two different temperatures.

Keywords: DNA synthesis(DNA合成), DNA labeling(DNA标记), DNA polymerization activity(DNA聚合活性), Pseudomonas(铜绿假单胞菌)

Materials and Reagents

  1. Pseudomonas syringae (P. syringae) Lz4W strain (Shivaji et al., 1989)
  2. Escherichia coli (E. coli) DH5alpha strain
  3. Random primer labelling kit (JONAKI Laboratory, catalog number: LCK 2 )
    Note: Many companies like Roche Diagnostics, Thermo Fisher Scientific, Agilent, Clontech etc. provide similar kit.
  4. Wizard Genomic DNA Purification Kit (Promega Corporation, catalog number: A1120 )
  5. (Alpha 32P)-dATP (JONAKI Laboratory, catalog number: LCP 103 )
  6. Lysozyme (Roche Diagnostics, catalog number: 10153516103 )
  7. 10% trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
  8. KCl (Sigma-Aldrich, catalog number: P9541 )
  9. 50 mM Tris.HCl (pH 7.5)
  10. 10% sucrose (Sigma-Aldrich, catalog number: 84097 )
  11. Peptone (HiMedia Laboratories, catalog number: RM 001-500G )
  12. Yeast extracts (HiMedia Laboratories, catalog number: RM 027-500G )
  13. Stop buffer (see Recipes)
  14. Lysozyme solution (see Recipes)
  15. ABM broth (Pavankumar et al., 2010) (see Recipes)

Equipment

  1. BD PrecisionGlide needles 22G (BD, catalog number: 305156 )
  2. Liquid scintillation analyzer (PerkinElmer, model: Tri-Carb 2900 TR )
  3. Centrifuge (Eppendorf, model: 5810R, catalog number: 5811000.010 )
  4. NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, catalog number 8482 )
  5. Micro centrifuge tubes (1.5 ml) (tarsons, catalog number: 500010 )
  6. F12- ED refrigerated/heating circulator (JULABO GmbH, catalog number: 9116612 )
  7. Plastic scintillation vials (PerkinElmer, catalog number: 6000477 )
  8. Separate laboratory design and workspace for radioactive work with proper guidelines

Procedure

  1. Preparation of cell extracts
    The basic method of cell extracts preparation was adapted from methods described earlier (Wickner et al., 1972; Villani et al., 1978).
    1. Inoculate P. syringae Lz4W strain from a single colony (streaked on ABM agar plate and was incubated at 22 °C for 24-36 h) in 5 ml ABM liquid media (autoclaved in 25 ml flask). Grow it for overnight at 22 °C shaking at 200 rpm.
    2. Inoculate a fresh secondary culture using the overnight grown cultures inoculum in a new 20 ml ABM broth (autoclaved in 100 ml flask) in 1:100 ratio dilution and grow at 22 °C till exponential growth phase (OD600~ 0.3 to 0.4, 6-7 h growth).
    3. Pellet down cells by centrifuging at 10,000 rpm for 5 min at 22 °C.
    4. Discard the supernatant, wash the pellet with 1 ml of buffer containing 10% sucrose containing 50 mM Tris.HCl (pH 7.5). Resuspend the pellet in 0.002 volume (40 µl for 20 ml culture) of 10% sucrose containing 50 mM Tris.HCl (pH 7.5) at room temperature (22-25 °C).
    5. Add 1/10 th volume (4 µl) of a lysozyme solution and 0.025 volume (1 µl) of 4 M KCl. Incubate the mixture at 0 °C (on ice) for 30 min.
    6. Centrifuge the lysate at 14,000 rpm for 45 min at 2 °C.
    7. Supernatant was transferred to a fresh microcentrifuge tube and stored at 0 °C (on ice). Quantify the total protein concentration using NanoDrop 1000 spectrophotometer.
      Note: Although this cell lysate is stable on ice for few hours, in most of our experiments we used this freshly prepared lysate within 30-60 min of preparation.

  2. Preparation of template DNA (E. coli genomic DNA)
    1. Isolate genomic DNA from E. coli strain MC4100 using genomic DNA isolation kit.
      Note: This step to be done one day before preparing the Pseudomonas cell extracts.
    2. Shear the genomic DNA by passing it through a large gauge (22G) needle for several times (15-16 times to get 6-10 kb size of sheared DNA).
    3. Determine the concentration of DNA by taking readings in NanoDrop 1000 spectrophotometer.
    4. Denature the template DNA by incubating it for 2-3 min in a boiling water bath, and then immediately freeze it into ice water bath. This DNA will be used as a template to assay the DNA polymerization activity using any Gram-negative bacterial cell extracts.

  3. DNA polymerization activity assay
    1. Pre-incubate the aliquots of Pseudomonas cell lysate at the desired temperatures for 10 min. We used 22 °C and 4 °C for P. syringae cell extracts.
    2. Prepare replicas (2 to 3) of two assay mixtures (experimental): One set to be incubated at 22 °C and another set at 4 °C.
    3. Each assay mixture (50 µl) will have 1 μg of heat denatured template DNA (sheared E. coli genomic DNA), Random primer buffer (1x), Random hexanucleotide primers (1x), 0.8 mM each of dCTP, dGTP, dTTP and 40 μCi of [alpha-32P]-dATP. Pre-incubate the samples at the experimental temperatures (22 °C and 4 °C) for at least 5 min.
      Note: Reaction volume to be adjusted using sterile Milli-Q water, considering the volume of cell extracts to be added, to make the final volume of 50 µl.
    4. Prepare two more sets of assay mixtures as controls and process them as above. Control (negative) assay mixtures will have everything except the template DNA.  
    5. In the mean time, label the tubes for collecting reaction samples at different time points. For example, for six time points, take 6 micro centrifuge tubes for each assay mixture and labeled them as 0, 15, 30, 60, 90 and 120 sec. Add 30 µl of E. coli unlabeled denatured DNA (50 µg)  in stop buffer and 40 µl of 10% TCA into each tube as stop buffer.
      Notes:
      1. Addition of E. coli unlabeled denatured DNA (cold DNA) increases the precipitation efficiency of labeled DNA (hot DNA).     
      2. The cold DNA was isolated from E. coli grown cells, heat denatured and immediately transferred in ice water.
    6. Now, to start the experiment, add 100 µg of pre-incubated cell lysates into the assay mixtures (step C4) which were incubated at 22 °C and 4 °C.
    7. Remove 10 µl of aliquote from each assay tubes at different time intervals (0, 15, 30, 60, 90 and 120 sec) and stop the reaction by adding into corresponding labeled tubes.
    8. Incubate these tubes for 1 h at 0 °C to precipitate the labeled DNA and then centrifuge at 14,000 rpm for 15 min at 4 °C.
    9. Remove the supernatant from the tubes containing unincorporated labels. To remove the drags, invert the tubes onto tissue paper or blotting paper for 2-3 min.
    10. Place centrifuge tubes into plastic scintillation vials and measure the radioactivity directly without adding any scintillation fluid (cherenkov counting) in a scintillation counter.
    11. Subtract the values (CPM) of control from the experimental value and calculate the rate of radioactivity incorporation.  
    12. The radioactivity (count per minute or CPM) incorporation into DNA or polymerization rate can be expressed as CPM per minute per milligram of protein in the cell extracts.

Representative data



Figure 1. DNA polymerization activity is higher at 22 °C compared to 4 °C. DNA polymerization activity was assessed using Pseudomonas cell extracts as described above. Incorporation of radioactive nucleotides was observed immediately after addition of cell lysate both at 22 °C and 4 °C. Activity reduced after 30 sec at 22 °C whereas in case of 4 °C reaction it went down after 60 sec. This decrease in radioactivity count could be due to higher activity of exonucleases present in the cell extracts. Therefore, polymerization rate should be calculated from the values obtained from the linear curves generated within 60 sec of experiment under the conditions. The maximum CPM achieved by cell lysate was used to calculate polymerization rate as CPM per minute.

Recipes

  1. Stop buffer
    10 mM Tris.HCl (pH 7.5)
    100 mM EDTA
  2. Lysozyme solution
    2 mg/ml in 0.025 M Tris.HCl (pH 7.6)
  3. ABM media
    0.5% peptone
    0.2% yeast extracts

Acknowledgments

The basic method of cell extracts preparation was adapted from methods described earlier (Wickner et al., 1972; Villani et al., 1978). The work was financed by a Grant from the Department of Science and Technology (DST), and by the Council of Scientific and Industrial Research (CSIR), Government of India.

References

  1. Pavankumar, T. L., Sinha, A. K. and Ray, M. K. (2010). All three subunits of RecBCD enzyme are essential for DNA repair and low-temperature growth in the Antarctic Pseudomonas syringae Lz4W. PloS One 5(2): e9412.
  2. Sinha, A. K., Pavankumar, T. L., Kamisetty, S., Mittal, P. and Ray, M. K. (2013). Replication arrest is a major threat to growth at low temperature in Antarctic Pseudomonas syringae Lz4W. Mol Microbiol 89(4): 792-810.
  3. Shivaji, S., Rao, N. S., Saisree, L., Sheth, V., Reddy, G. S. and Bhargava, P. M. (1989). Isolation and identification of Pseudomonas spp. from Schirmacher Oasis, Antarctica. Appl Environ Microbiol 55(3): 767-770.
  4. Villani, G., Boiteux, S. and Radman, M. (1978). Mechanism of ultraviolet-induced mutagenesis: extent and fidelity of in vitro DNA synthesis on irradiated templates. Proc Natl Acad Sci U S A 75(7): 3037-3041.
  5. Wickner, R. B., Wright, M., Wickner, S. and Hurwitz, J. (1972). Conversion of phiX174 and fd single-stranded DNA to replicative forms in extracts of Escherichia coli. Proc Natl Acad Sci U S A 69(11): 3233-3237.

简介

该方案已经设计用于测量南极细菌假单胞菌syrinagae Lz4W的粗细胞提取物中的体外 DNA聚合活性。 该细菌可以在4℃下生长,在22℃以最佳生长速率生长。 在低温(4℃)下观察到的细菌与较高温度(22℃)相比的缓慢生长速率可以归因于在低温下DNA复制的速率降低。 在这里我们描述了一个协议,我们已经用来量化的体外细胞提取物在两个不同的温度下的DNA聚合。

关键字:DNA合成, DNA标记, DNA聚合活性, 铜绿假单胞菌

材料和试剂

  1. < em>丁香假单胞菌(< em>丁香水杨)Lz4W菌株(Shivaji等人,1989)
  2. 大肠杆菌(大肠杆菌)DH5α菌株
  3. 随机引物标记试剂盒(JONAKI Laboratory,目录号:LCK 2)
    注意:许多公司如Roche Diagnostics,Thermo Fisher Scientific,Agilent,Clontech等都提供类似的工具包。
  4. Wizard基因组DNA纯化试剂盒(Promega Corporation,目录号:A1120)
  5. (Alpha <32> P)-dATP(JONAKI Laboratory,目录号:LCP 103)
  6. 溶菌酶(Roche Diagnostics,目录号:10153516103)
  7. 10%三氯乙酸(TCA)(Sigma-Aldrich,目录号:T6399)
  8. KCl(Sigma-Aldrich,目录号:P9541)
  9. 50mM Tris(pH7.5)
  10. 10%蔗糖(Sigma-Aldrich,目录号:84097)
  11. 蛋白胨(HiMedia Laboratories,目录号:RM 001-500G)
  12. 酵母提取物(HiMedia Laboratories,目录号:RM 027-500G)
  13. 停止缓冲区(参见配方)
  14. 溶菌酶溶液(参见配方)
  15. ABM肉汤(Pavankumar等人,2010)(参见Recipes)

设备

  1. BD PrecisionGlide针22G(BD,目录号:305156)
  2. 液体闪烁分析仪(PerkinElmer,型号:Tri-Carb 2900 TR)
  3. 离心机(Eppendorf,型号:5810R,目录号:5811000.010)
  4. NanoDrop 1000分光光度计(Thermo Fisher Scientific,目录号8482)
  5. 微量离心管(1.5ml)(tarsons,目录号:500010)
  6. F12-ED制冷/加热循环器(JULABO GmbH,目录号:9116612)
  7. 塑料闪烁瓶(PerkinElmer,目录号:6000477)
  8. 使用适当的指南分离放射性工作的实验室设计和工作空间

程序

  1. 细胞提取物的制备
    细胞提取物制备的基本方法改编自早期描述的方法(Wickner等人,1972; Villani等人,1978)。
    1. 接种 P。 (在25ml烧瓶中高压灭菌)中的单个菌落(在ABM琼脂平板上划线并在22℃下温育24-36小时)中培养大肠杆菌Lz4W菌株。 在22℃下以200rpm振荡生长过夜。
    2. 使用过夜生长的培养物接种物在新的20ml ABM肉汤(在100ml烧瓶中高压灭菌)中以1:100比例稀释度接种新鲜的次级培养物,并在22℃下生长直至指数生长期(OD <600μm) >〜0.3〜0.4,生长6-7小时)
    3. 通过在22℃下以10,000rpm离心5分钟沉淀细胞
    4. 弃去上清液,用1ml含有10%含有50mM Tris-盐酸(pH7.5)的蔗糖的缓冲液洗涤沉淀。在室温(22-25℃)下,将0.002体积(40μl,对于20ml培养物)的10%蔗糖重悬于含有50mM Tris缓冲液(pH7.5)的沉淀中。
    5. 加入1/10体积(4μl)溶菌酶溶液和0.025体积(1μl)4M KCl。在0℃(在冰上)孵育混合物30分钟。
    6. 在2℃下以14,000rpm离心裂解物45分钟
    7. 将上清液转移到新鲜的微量离心管中并在0℃(在冰上)储存。使用NanoDrop 1000分光光度计量化总蛋白浓度。
      注意:虽然这种细胞裂解物在冰上稳定几个小时,但在我们的大多数实验中,我们在制备的30-60分钟内使用这种新鲜制备的裂解物。

  2. 模板DNA(大肠杆菌基因组DNA)的制备
    1. 使用基因组DNA分离试剂盒从大肠杆菌菌株MC4100分离基因组DNA 注意:这个步骤要在准备假单胞菌细胞提取物前一天进行。
    2. 通过使其通过大规格(22G)针几次(15-16次以获得6-10kb大小的剪切的DNA)剪切基因组DNA。
    3. 通过在NanoDrop 1000分光光度计中读取读数来确定DNA的浓度
    4. 通过在沸水浴中孵育2-3分钟来使模板DNA变性,然后立即将其冻结到冰水浴中。 该DNA将用作模板,以使用任何革兰氏阴性细菌细胞提取物测定DNA聚合活性

  3. DNA聚合活性测定
    1. 在所需温度下预温育假单胞菌细胞裂解物的等分试样10分钟。我们对于P使用22℃和4℃。丁香液细胞提取物
    2. 制备两种测定混合物(实验)的复制品(2至3):一组在22℃下培养,另一组在4℃下培养。
    3. 每种测定混合物(50μl)将具有1μg热变性模板DNA(剪切的大肠杆菌基因组DNA),随机引物缓冲液(1x),随机六核苷酸引物(1x),每种0.8mM dCTP,dGTP,dTTP和40μCi的[α-32P] -dATP。在实验温度(22℃和4℃)预孵育样品至少5分钟。
      注意:使用无菌Milli-Q水调节反应体积,考虑要添加的细胞提取物的体积,使最终体积为50μl。
    4. 准备另外两套测定混合物作为对照,并如上所述进行处理。对照(阴性)测定混合物将具有除模板DNA之外的所有物质。  
    5. 同时,标记用于在不同时间点收集反应样品的管。例如,对于6个时间点,对于每个测定混合物取6个微量离心管,并将其标记为0,15,30,60,90和120秒。加入30微升的E。大肠杆菌未标记的变性DNA(50μg)在终止缓冲液中,并将40μl10%TCA加入每个管中作为终止缓冲液 注意:
      1. <大肠杆菌未标记的变性DNA(冷DNA)的添加增加了标记的DNA(热DNA)的沉淀效率。    
      2. 冷的DNA从大肠杆菌生长的细胞中分离,热变性并立即转移到冰水中。
    6. 现在,为了开始实验,将100μg预孵育的细胞裂解物加入在22℃和4℃孵育的测定混合物(步骤C4)中。
    7. 从不同的时间间隔(0,15,30,60,90和120秒)从每个测定管中取出10微升等分试样,通过加入相应的标记管停止反应。
    8. 在0℃下孵育这些管1小时以沉淀标记的DNA,然后在4℃下以14,000rpm离心15分钟。
    9. 从含有未掺入标记物的试管中除去上清液。 为了除去药物,将管倒入薄纸或吸墨纸上2-3分钟。
    10. 将离心管放入塑料闪烁瓶中,直接测量放射性,而不在闪烁计数器中加入任何闪烁液(cherenkov计数)。
    11. 从实验值中减去对照的值(CPM),并计算放射性掺入率。  
    12. 放射性(每分钟计数或CPM)或DNA聚合速率可以表示为细胞提取物中每毫克蛋白质的每分钟CPM。
  • 在0℃下孵育这些管1小时以沉淀标记的DNA,然后在4℃下以14,000rpm离心15分钟。
  • 从含有未掺入标记物的试管中除去上清液。 为了除去药物,将管倒入薄纸或吸墨纸上2-3分钟。
  • 将离心管放入塑料闪烁瓶中,直接测量放射性,而不在闪烁计数器中加入任何闪烁液(cherenkov计数)。
  • 从实验值中减去对照的值(CPM),并计算放射性掺入率。  
  • 放射性(每分钟计数或CPM)或DNA聚合速率可以表示为细胞提取物中每毫克蛋白质的每分钟CPM。
  • ...
  • Lysozyme solution
    2 mg/ml in 0.025 M Tris.HCl (pH 7.6)
  • ABM media
    0.5% peptone
    0.2% yeast extracts
  • Acknowledgments

    The basic method of cell extracts preparation was adapted from methods described earlier (Wickner et al., 1972; Villani et al., 1978). The work was financed by a Grant from the Department of Science and Technology (DST), and by the Council of Scientific and Industrial Research (CSIR), Government of India.

    参考文献

    1. Pavankumar,T.L.,Sinha,A.K.and Ray,M.K。(2010)。 RecBCD酶的所有三个亚基都是对于南极假单胞菌(Pseudomonas syringae)Ls4W的DNA修复和低温生长至关重要。

      5(2):e9412。
    2. Sinha,A.K.,Pavankumar,T.L.,Kamisetty,S.,Mittal,P.and Ray,M.K。(2013)。 复制停止是南极地区低温生长的主要威胁伪狂犬病毒/em> Lz4W。 Mol Microbiol 89(4):792-810。
    3. Shivaji,S.,Rao,N.S.,Saisree,L.,Sheth,V.,Reddy,G.S。和Bhargava,P.M。(1989)。 假单胞菌的分离和鉴定。从 ,Appl。Environ Microbiol 55(3):767-770。
    4. Villani,G.,Boiteux,S。和Radman,M。(1978)。 紫外线诱导诱变的机制:体外细胞的程度和保真度 DNA在辐射模板上的合成。美国国家科学院院刊75(7):3037-3041。
    5. Wickner,R.B.,Wright,M.,Wickner,S。和Hurwitz,J。(1972)。 将phiX174和fd单链DNA转化为大肠杆菌提取物中的复制型/em>。美国国家科学院院报69(11):3233-3237。
    • English
    • 中文翻译
    免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
    引用:Sinha, A. K. and Ray, M. K. (2014). In vitro DNA Polymerization Activity Assay Using Cell-free Extracts. Bio-protocol 4(16): e1207. DOI: 10.21769/BioProtoc.1207.
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