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Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg2+ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg2+. The relative fidelity of the polymerase can then be estimated using the standard extension efficiency. The assay described here is based on the method published in Mendelman et al. (1990).

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Mismatched Primer Extension Assays
错配引物延伸法

微生物学 > 微生物生物化学 > 蛋白质 > 活性
作者: Vasudevan Achuthan
Vasudevan AchuthanAffiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA
Bio-protocol author page: a2310
 and Jeffrey J. DeStefano
Jeffrey J. DeStefanoAffiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA
For correspondence: jdestefa@umd.edu
Bio-protocol author page: a2311
Vol 5, Iss 12, 6/20/2015, 1816 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1508

[Abstract] Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg2+ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg2+. The relative fidelity of the polymerase can then be estimated using the standard extension efficiency. The assay described here is based on the method published in Mendelman et al. (1990).

[Abstract] 稳态动力学测定法是估计几种聚合酶的保真度的可靠方法(Menendez-Arias,2009; Rezende和Prasad,2004; Svarovskaia等人,2003)。分析具有在3'末端的特异性错配的引物的延伸的能力是错配引物延伸测定的主要强度。最近,我们使用错配的引物延伸测定法显示当Mg 2+浓度降低到生理相关浓度(〜0.25mM)时,HIV RT的保真度显着增加(Achuthan等al 。,2014)。在这里,我们详细地描述如何进行错配引物延伸测定以使用人类免疫缺陷病毒逆转录酶(HIV RT)在2mM Mg 2+ 2 + 测量标准延伸效率。然后可以使用标准延伸效率估计聚合酶的相对保真度。这里描述的测定基于在Mendelman等人(1990)中公开的方法。

Materials and Reagents

  1. Deoxynucleoside triphosphate (Roche Diagnostics, catalog number: 11969064001 )
  2. Gamma [γ-32P] ATP (PerkinElmer, catalog number: Blu502A001MC )
  3. G-25 Macro spin columns (best suited for volumes of 75-150 μl) (Harvard Apparatus, catalog number: 74-3901 )
  4. 40% Acrylamide-Bisacrylamide (19:1) solution (VWR International, catalog number: JT4969-0 )
  5. T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number: M0201L )
  6. 10X T4 polynucleotide kinase buffer (New England Biolabs, catalog number: B0201S )
  7. Urea (VWR International, catalog number: 97061-926 )
  8. Ammonium Persulfate (VWR International, catalog number: 97064-594 )
  9. HIV Reverse Transcriptase, purified as described in Hou et al. (2004) 
  10. DNA oligonucleotides from Integrated DNA Technologies
    1. Template:
      5'-GGGCGAATTTAG[G/C]TTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTG G-3’. The underlined nucleotides in brackets indicate that templates with either a G or C at this position can be used depending on the type of mismatch examined.
    2. Primer:
      5ʹ-TAACCCTCACTAAAGGGAACAAAAX-3ʹ. “X” at the 3ʹ end of the primer denotes A, T, or C depending on the mismatch examined. “X” in the case of a matched primer is G.
  11. 1 M MgCl2
  12. Extension reaction buffer (see Recipes)
  13. 2x loading dye (see Recipes)

Equipment

  1. Eppendorf tubes
  2. Micropipette
  3. Table top centrifuge
  4. Incubator
  5. Gel apparatus

Software

  1. Sigmaplot Version 10.0 (Sysstat Software)

Procedure

  1. Primer labelling
    1. All the primers should be first radiolabelled in 50 µl of 1x PNK buffer along with 50 pico moles of each primer, 10 μl of [γ-32P] ATP and 5 units of PNK.
      Note: The reaction mixture was incubated for 30 min at 37 °C and the PNK was heat inactivated for 15 min at 65 °C.
    2. G-25 spin columns were incubated with 500 µl dH2O for 15 min to equilibrate the column and the water was removed by spinning the columns at a table top centrifuge at 5,000 rpm for 4 min.
    3. After heat inactivation, the excess [γ-32P] ATP was removed from the reaction mixture by loading it onto an equilibrated column and spinning at 5,000 rpm for 4 min.
  2. Matched primer extension reactions
    To obtain information about the standard extension efficiency, extension of matched as well as mismatched primers should be performed.  The standard extension efficiency can then be calculated as the ratio of efficiency of extending mismatched primers to efficiency of extending matched primers.
    1. Eight matched primer extension reactions were set up. For each reaction, 14 nM of the radiolabeled primer was hybridized to 14 nM of the template (1:1 ratio of primer:template) in 7 μl of the extension reaction buffer. The mixture was heated at 65 °C for 5 min and then slowly cooled to room temperature.
    2. The hybrid was then incubated for 3 min at 37 °C in the reaction buffer along with 2 μl of 10 mM MgCl2 (final concentration of 2 mM MgCl2) and 2 μl of the nucleotide substrate (concentration varies for each reaction, see below) for each reactions. The nucleotide substrate is the next correct nucleotide to be added and it depends on the template used in the reactions.  For this particular template, dCTP was the substrate (Figure 1). For matched primer extension reactions, the eight reactions had a final concentration of dCTP in the order of 0, 0.02, 0.04, 0.1, 0.2, 0.3, 0.6 and 1 μM respectively.


      Figure 1. Constructs used in mismatched primer extension assays. The sequence of the DNA templates is shown at the bottom and the sequence of the primer at the top. The underlined nucleotides show the only differences between the two templates.

    3. The extension was then initiated by addition of 2 μl of 13 nM HIV RT (2 nM final concentration). The total reaction volume was 13 μl.
    4. After 2 min, reactions were terminated by addition of 13 μl of 2x loading dye.
      Note: Reactions were run only for 2 min to ensure the primer is extended by only one nucleotide.
    5. The reaction products were then electrophoresed on 16% denaturing 7 M urea-polyacrylamide gels, dried, and imaged using a Fujifilm FLA5100 phosphorimager.
      Note: The samples were run far enough to separate the extended band from the primer band (Figure 2).
  3. Mismatched primer extension reactions
    1. For mismatched primer extension reactions, a different radiolabeled primer, depending on the mismatch analyzed (Figure 1), is used. Primer-template hybrids were made as described above.
    2. Eight individual reactions were set up. 7 μl of primer-template hybrids was incubated at 37 °C in the reaction buffer for 3 min along with 2 μl of 10 mM MgCl2 (final concentration of 2 mM MgCl2) and 2 μl of the nucleotide substrate. The total reaction volume was 13 μl.
      Note: Mismatched primer-template sequences require more substrate for extension than matched primer-template sequences. So, the eight reactions had a final concentration of dCTP in the order of 0, 50, 100, 200, 400, 630, 1,200 and 1,870 μM respectively (Figure 2).
    3. Extension was initiated by addition of 2 μl of 13 nM HIV RT.
    4. After 5 min of extension, the reactions were terminated by addition of 13 μl of 2x loading dye and the extension products were processed on a 16% denaturing polyacrylamide gel as described above. The gel was run at 75 Watts for 90 min.


      Figure 2. Representative data for the mismatched primer extension assay. Primer: template hybrid used here had a C.A mismatch at the 3ʹ terminus. Concentration of dCTP used in each of the lanes (left to right): 0, 50, 100, 200, 400, 630, 1,200 and 1,870 μM.

  4. Calculation of standard extension efficiency
    1. Velocity measurements were performed according to Mendelman et al. (1990). Velocity (rel) of extending each primer by one nucleotide was calculated according to the formula:  , where is the intensity of the extended band,  is the intensity of the primer band, and t is the time of extension.
    2. rel (percentage of primer extended per minute) was then plotted against [S], where [S] is the concentration of the substrate (dCTP) used in the reaction (Figure 3).  
      Note: These curves are typical of Michaelis-Menten kinetics curves.
    3. The values of Vmax and Km were obtained by curve-fitting. The data were fitted to a “Single rectangular 2-parameter hyperbola equation” using the Sigmaplot software.
    4. The constant “a” obtained after curve fitting corresponds to the Vmax and “b” corresponds to the Km for this extension. Vmax and Km values were individually determined for both matched and mismatched primer extension reactions.
    5. The standard extension efficiency,  , was calculated as the ratio of  .


      Figure 3. Representative graph for the mismatched primer extension assay. Primer:template hybrid used here had a C.A mismatch at the 3ʹ terminus. Vmax and Km were determined by fitting the points to a “Single rectangular 2-parameter hyperbola equation” using the Sigmaplot software.

Recipes

  1. Extension reaction buffer (50 ml)
    1 M Tris HCl (pH 8)
    25 ml
    3 M KCl
    13.3 ml
    1 M DTT
    1 ml
    RNase free water
    10.7 ml
  2. 2x loading dye (10 ml)
    50 mM Tris HCl (pH 6.8)
    500 μl
    100 mM DTT
    1 ml
    2% SDS
    2 ml
    0.05% Bromophenol blue
    500 μl
    10% glycerol
    1 ml
    RNase free water
    5 ml

References

  1. Achuthan, V., Keith, B. J., Connolly, B. A. and DeStefano, J. J. (2014). Human immunodeficiency virus reverse transcriptase displays dramatically higher fidelity under physiological magnesium conditions in vitro. J Virol 88(15): 8514-8527.
  2. Hou, E. W., Prasad, R., Beard, W. A. and Wilson, S. H. (2004). High-level expression and purification of untagged and histidine-tagged HIV-1 reverse transcriptase. Protein Expr Purif 34(1): 75-86.
  3. Mendelman, L. V., Petruska, J. and Goodman, M. F. (1990). Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. J Biol Chem 265(4): 2338-2346.
  4. Menendez-Arias, L. (2009). Mutation rates and intrinsic fidelity of retroviral reverse transcriptases. Viruses 1(3): 1137-1165.
  5. Rezende, L. F. and Prasad, V. R. (2004). Nucleoside-analog resistance mutations in HIV-1 reverse transcriptase and their influence on polymerase fidelity and viral mutation rates. Int J Biochem Cell Biol 36(9): 1716-1734.
  6. Svarovskaia, E. S., Cheslock, S. R., Zhang, W. H., Hu, W. S. and Pathak, V. K. (2003). Retroviral mutation rates and reverse transcriptase fidelity. Front Biosci 8: d117-134.

材料和试剂

  1. 脱氧核苷三磷酸(Roche Diagnostics,目录号:11969064001)
  2. γ-[sup-32P] ATP(PerkinElmer,目录号:Blu502A001MC)
  3. G-25宏旋转柱(最适合体积为75-150μl)(哈佛仪器,目录号:74-3901)
  4. 40%丙烯酰胺 - 双丙烯酰胺(19:1)溶液(VWR International,目录号:JT4969-0)
  5. T4多核苷酸激酶(PNK)(New England Biolabs,目录号:M0201L)
  6. 10X T4多核苷酸激酶缓冲液(New England Biolabs,目录号:B0201S)
  7. 尿素(VWR International,目录号:97061-926)
  8. 过硫酸铵(VWR International,目录号:97064-594)
  9. HIV逆转录酶,如Hou等人(2004)所述纯化,
  10. DNA DNA寡核苷酸
    1. 模板:
      5'-GGGCGAATTTAG [ G/C TTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTG   G-3'。 括号中带下划线的核苷酸表示模板 在该位置可以使用G或C,这取决于类型 的错配。
    2. 引子:
      5'-TAACCCTCACTAAAGGGAACAAAAX-3'。   引物3'端的"X"表示A,T或C. 失配检查。 在匹配引物的情况下,"X"是G.
  11. 1 M MgCl 2
  12. 延伸反应缓冲液(参见配方)
  13. 2x加载染料(参见配方)

设备

  1. Eppendorf管
  2. 微量移液器
  3. 台式离心机
  4. 孵化器
  5. 凝胶装置

软件

  1. Sigmaplot版本10.0(Sysstat软件)

程序

  1. 引物标记
    1. 所有引物应首先在50μl的1×PNK缓冲液中以及50皮摩尔每种引物,10μl[γ-32 P] ATP和5单位PNK进行放射性标记。
      注意:将反应混合物在37℃下孵育30分钟,并将PNK在65℃下热灭活15分钟。
    2. 将G-25离心柱与500μldH 2 O孵育15分钟以平衡柱,并且通过在台式离心机中以5,000rpm离心4分钟来除去水。
    3. 在热失活后,通过将其装载到平衡的柱上并以5,000rpm旋转4分钟,从反应混合物中除去过量的[γ-32P] ATP。
  2. 匹配引物延伸反应
    要获得有关标准延伸效率的信息,应进行匹配引物和错配引物的延伸。然后标准延伸效率可以计算为延伸错配引物的效率与延伸匹配引物的效率的比率
    1. 建立了8个匹配的引物延伸反应。对于每个反应,将14nM的放射性标记的引物与14nM的模板(引物:模板的1:1比例)在7μl的延伸反应缓冲液中杂交。将混合物在65℃下加热5分钟,然后缓慢冷却至室温。
    2. 然后将杂交体与2μl的10mM MgCl 2(终浓度为2mM MgCl 2)和2μl的10mM MgCl 2溶液一起在37℃下在反应缓冲液中孵育3分钟。 μl的核苷酸底物(每种反应的浓度不同,见下文)。核苷酸底物是待添加的下一个正确核苷酸,并且其取决于反应中使用的模板。对于该特定模板,dCTP是底物(图1)。对于匹配的引物延伸反应,八个反应的dCTP终浓度分别为0,0.02,0.04,0.1,0.2,0.3,0.6和1μM。


      图1.在错配引物延伸测定中使用的构建体。 DNA模板的序列显示在底部,引物的序列显示在顶部。加下划线的核苷酸显示两个模板之间的唯一区别。

    3. 然后通过加入2μl13nM HIV RT(2nM终浓度)引发延伸。总反应体积为13μl。
    4. 2分钟后,通过加入13μl2x加载染料终止反应 注意:反应仅进行2分钟以确保引物仅延伸一个核苷酸。
    5. 然后将反应产物在16%变性7M尿素 - 聚丙烯酰胺凝胶上电泳,干燥,并使用Fujifilm FLA5100磷光计成像。
      注意:样品运行足够远,以从引物条带中分离扩展条带(图2)。
  3. 引物延伸反应不匹配
    1. 对于错配的引物延伸反应,使用不同的放射性标记的引物,这取决于分析的错配(图1)。如上所述制备引物模板杂交体。
    2. 建立了八个单独的反应。将7μl引物 - 模板杂交体在37℃下在反应缓冲液中与2μl10mM MgCl 2(终浓度为2mM MgCl 2)一起温育3分钟>)和2μl核苷酸底物。总反应体积为13μl 注意:不匹配的引物 - 模板序列比匹配的引物 - 模板序列需要更多的延伸底物。因此,八个反应的dCTP最终浓度分别为0,50,100,200,400,630,1,200和1,870μM(图2)。
    3. 通过加入2μl13nM HIV RT启动延伸。
    4. 延伸5分钟后,通过加入13μl2x加载染料终止反应,并且如上所述在16%变性聚丙烯酰胺凝胶上处理延伸产物。凝胶在75瓦下运行90分钟。


      图2.错配引物延伸测定的代表性数据。 此处使用的引物:模板杂交体在3'末端具有C.A错配。在每个泳道中使用的dCTP的浓度(从左到右):0,50,100,200,400,630,1,200和1,870μM。

  4. 计算标准延伸效率
    1. 根据Mendelman等人(1990)进行速度测量。将每个引物延伸一个核苷酸的速度( 根据以下公式计算: ,其中  是扩展频带的强度,是引物条带的强度,t是延伸的时间。
    2. 然后将 (引物每分钟延伸的百分比)对[S]作图,其中[S]是反应中使用的底物(dCTP)的浓度(图3)。  
      注意:这些曲线是Michaelis-Menten动力学曲线的典型曲线。
    3. 通过曲线拟合获得V max和K m的值。使用Sigmaplot软件将数据拟合到"单矩形2-参数双曲线方程"。
    4. 在曲线拟合之后获得的常数"a"对应于V sub max,并且"b"对应于该延伸的K sub。对匹配和错配引物延伸反应分别测定V max和K m值。
    5. 标准扩展效率  被计算为


      图3.错配引物延伸测定的代表图。在此使用的引物:模板杂交体在3'末端具有C.A错配。通过将点拟合为"单一"来确定Vmax和Km 矩形2参数双曲线方程"使用Sigmaplot软件。

食谱

  1. 延伸反应缓冲液(50ml)
    1 M Tris HCl(pH 8)
    25 ml
    3 M KCl
    13.3毫升
    1 M DTT
    1 ml
    无RNase水
    10.7 ml
  2. 2x加载染料(10ml)
    50mM Tris HCl(pH6.8)
    500微升
    100 mM DTT
    1 ml
    2%SDS
    2 ml
    0.05%溴酚蓝色
    500微升
    10%甘油 1 ml
    无RNase水
    5 ml

参考文献

  1. Achuthan,V.,Keith,B.J.,Connolly,B.A。和DeStefano,J.J。(2014)。 人体免疫缺陷病毒逆转录酶在vitr中在生理性镁条件下显示出更高的保真度 > 。 J Virol 88(15):8514-8527。
  2. Hou,E.W.,Prasad,R.,Beard,W.A。和Wilson,S.H。(2004)。 未标记和组氨酸标记的HIV-1反向的高水平表达和纯化 蛋白质表达纯化 34(1):75-86。
  3. Mendelman,L.V.,Petruska,J。和Goodman,M.F。(1990)。 基本错配扩展动力学。 DNA polymerase alpha和reverse transcriptase的比较。"生物化学杂志"265(4):2338-2346。
  4. Menendez-Arias,L。(2009)。 逆转录病毒逆转录酶的突变率和内在保真度 病毒 1(3):1137-1165。
  5. Rezende,L.F.and Prasad,V.R。(2004)。 HIV-1逆转录酶中的核苷类似物抗性突变及其对聚合酶保真度和病毒突变率的影响。 Int J Biochem Cell Biol 36(9):1716-1734。
  6. Svarovskaia,E.S.,Cheslock,S.R.,Zhang,W.H.,Hu,W.S.and Pathak,V.K。(2003)。 逆转录病毒突变率和逆转录酶忠诚度。 Front Biosci 8:d117-134。
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How to cite this protocol: Achuthan, V. and DeStefano, J. J. (2015). Mismatched Primer Extension Assays. Bio-protocol 5(12): e1508. DOI: 10.21769/BioProtoc.1508; Full Text



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