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Primer Extension Reactions for the PCR- based α- complementation Assay
引物延伸反应用于基于PCR的α-互补试验   

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Abstract

The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.

Materials and Reagents

  1. pBSM13+ (Stratagene)
  2. T3 RNA polymerase (Roche Diagnostics, catalog number: P2083 )
  3. 10x transcription buffer (Roche Diagnostics, catalog number: P2083 )
  4. High-fidelity PvuII (PvuII-HF) (New England Biolabs, catalog number: R3151L )
  5. High-fidelity EcoRI (EcoRI-HF) (New England Biolabs, catalog number: R3101L )
  6. 10x CutSmart buffer (New England Biolabs, catalog number: R3101L)
  7. 6x gel loading dye (New England Biolabs, catalog number: R3101L)
  8. NdeI (New England Biolabs, catalog number: R0111L )
  9. T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number: M0201L )
  10. 10x T4 polynucleotide kinase buffer (New England Biolabs, catalog Number: B0201S )
  11. RNasin (RNase inhibitor) (New England Biolabs, catalog number: M0307L )
  12. RNase (DNase-free) (Roche Diagnostics, catalog number: 11119915001 )
  13. RNase-free DNase I (Affymetrix, catalog number: 784111000 )
  14. Pfu DNA polymerase (Agilent Technologies, catalog number: 600353 )
  15. 10x Pfu buffer (Agilent Technologies, catalog number: 600353)
  16. Ribonucleoside triphosphate set (Roche Diagnostics, catalog number: 11277057001 )
  17. Deoxynucleoside triphosphate (dNTP) (Roche Diagnostics, catalog number: 11969064001 )
  18. Gamma [γ-32P] ATP (PerkinElmer, catalog number: Blu502A001MC )
  19. G-25 Macro spin columns (best suited for volumes of 75-150 μl) (Harvard Apparatus, catalog number: 74-3901 )
  20. RNeasy RNA purification kit (QIAGEN, catalog number: 74104 )
  21. Phenol: Chloroform: Isoamyl alcohol (25:24:1) (Amresco, catalog number: K169-400ML )
  22. Ethanol (VWR Lifesciences, catalog number: EM1.00967.4003 )
  23. 3M Sodium Acetate (Amresco, catalog number: E521-100ML )
  24. Isopropyl alcohol (J.T.Baker®, catalog number: 9037-03 )
  25. 40% Acrylamide-Bisacrylamide (19:1) solution (VWR International, catalog number: JT4968-0 )
  26. 40% Acrylamide-Bisacrylamide (29:1) solution (VWR International, catalog number: JT4968-0 )
  27. Urea (VWR International, catalog number: 97061-926 )
  28. Ammonium Persulfate (VWR International, catalog number: 97064-594 )
  29. HIV Reverse Transcriptase, [purified as described in Hou et al. (2004)]
  30. Milli-Q quality [RNase, DNase free water (dH2O)]
  31. DNA oligonucleotides were obtained from Integrated DNA Technologies
  32. Extension reaction buffer (see Recipes)
  33. Elution buffer (see Recipes)
  34. 2x SDS loading buffer (see Recipes)

Equipment

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

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 polynucleotide kinase (PNK). 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 excess 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. Preparation of RNA for fidelity assay
    1. The transcript used as a template for the fidelity assay was derived from the plasmid pBSΔPvuII1146, which was prepared as described in DeStefano et al. (1998).
    2. For preparation of the RNA, 10 μg of the plasmid was cleaved with 50 units of the enzyme NdeI in the NEB buffer 4 for 3 h at 37 °C. The cleaved plasmid was then extracted with phenol chloroform extraction and recovered by ethanol precipitation as described below.
    3. After cleavage, run-off transcription was performed in 100 µl of the transcription buffer along with 2 µg of the linearized plasmid, 5 µl of 100 mM DTT, 10 µl of 5 mM ribonucleotides, 2 µl of RNase inhibitor, and 40 units of T3 RNA polymerase for 3 h at 37 °C.
    4. 10 units of DNase I was added to the reaction mixture and the reaction was incubated for 10 min to digest the remaining DNA.
    5. The RNA was then purified using the QIAGEN RNeasy kit, as per the manufacturer’s instructions, and quantified using the spectrophotometer.

  3. RNA-directed DNA synthesis
    1. The ~760 nt RNA template, prepared using the method described above, was hybridized to a radiolabeled 25-nt DNA primer (5ʹ-GCGGGCCTCTTCGCTATTACGCCAG-3ʹ).
    2. For hybridization, 50 nM of the primer was added to 25 nM of the template (2:1 ratio of primer: template) in 48 μl of the extension reaction buffer along with 6 mM MgCl2 and 100 μM dNTPs. The mixture was heated at 65 °C for 5 min and then slowly cooled to room temperature. The total reaction volume was 50 μl.
    3. The primer- template was incubated at 37 °C for 3 min. 2 μl of 5 μM HIV RT was added to initiate the extension reaction and the incubation was continued for 30 min. Full extension of the primer should yield a 199 nucleotide (nt) DNA product (Figure 1).
      Note: The primer used here was diluted 10-fold with unlabeled primer so that the extension product from this round will have less specific radioactivity than the product from the next round of synthesis (Figure 2).


      Figure 1. Schematic illustration of the assay. RNAs are indicated by broken lines and DNAs by solid lines. Primers have arrowheads at the 3ʹ end. The ~760-nucleotide template RNA used as the initial template for HIV RT RNA-directed DNA synthesis is shown at the top with the 3ʹ and 5ʹ ends indicated. The positions of PvuII and EcoRI restriction sites are indicated for reference to the vector. Numbering is based on that for the plasmid pBSM13+.


      Figure 2. Representative Data after two rounds of synthesis. Products from the DNA-directed synthesis appear darker in the gel than the products from the first round because the primers used for this step had higher specific activity.

    4. After 30 min, 1 μl of RNase was added to digest the remaining RNA and the sample was heated to 65 °C for 5 min to deactivate the RT.
    5. The DNA product was then recovered by standard phenol chloroform extraction. Equal volume (50 μl) of phenol: chloroform: isoamyl alcohol mixture (25:24:1) was added to the reaction. The reaction was mixed well and then spun in the microcentrifuge at 18,000 rpm for 3 min. The aqueous layer from the top was removed, transferred to a new tube and equal volume (~50 μl) of chloroform was added. The mixture was spun again at 18,000 rpm for 3 min in the microcentrifuge and the aqueous layer was removed carefully and moved to a new tube.
    6. DNA product was precipitated using standard ethanol precipitation. Two volumes of 100% ethanol (100 μl) and one-tenth volume (5 μl) of 3M sodium acetate were added and the reaction was stored at -20 °C for two hours.
    7. After two hours, the reaction was spun for 20 min at 18,000 rpm. The supernatant was discarded carefully. The pellet was washed with 500 µl of 70% ethanol and then dried.
    8. The pellet was resuspended in 2x loading buffer and the products were separated on a 6% denaturing 7 M urea-polyacrylamide gels.
    9. 199 nt DNA product was cut out from the gel and the gel pieces was crushed and re-suspended in 500 μl elution buffer. After overnight elution at 4 °C, the material was filtered through 0.45 μM syringe filter and the DNA product was recovered using phenol chloroform extraction method described above.
    10. The recovered product was resuspended in elution buffer, quantified using a spectrophotometer, and used as a template for the DNA directed DNA synthesis step.

  4. DNA directed DNA synthesis
    1. The recovered DNA product from RNA directed synthesis was hybridized as described above to another 20-nt radiolabeled DNA primer (5ʹ-AGGATCCCCGGGTACCGAGC-3ʹ) with at least 10-fold-greater specific activity than the primer used for round 1 (see above).
    2. A second round of DNA synthesis was performed as described above except that the reaction volume was 25 μl. Conditions for extension were maintained identical in the RNA and DNA template extension reactions.
    3. Reactions were terminated with 25 µl of 2x loading buffer, and products were gel purified on an 8% denaturing 7 M urea-polyacrylamide gels.
      Note: The gel was run far enough to efficiently separate the 162-nt full extension product of round 2 from the 199-nt template (Figure 2).
    4. The 162 nt product was located and cut out from the gel. The product was recovered as described above and resuspended in 100 μl of elution buffer.

  5. PCR reactions
    1. The round 2 DNA produced in the above-described step was amplified by PCR using the following primers: 5ʹ-GCGGGCCTCTTCGCTATTACGCCAG-3ʹ and 5ʹ-AGGATCCCCGGGTACCGAGC-3ʹ.
    2. The first primer is the one used for the RNA directed DNA synthesis and the second primer overlaps a region of the EcoRI site on the plasmid from which the RNA was originally transcribed.
    3. PCR reactions were performed in the purchased 1x Pfu buffer along with 200 μM dNTPs, 50 pico moles of each primer, and 5 units of Pfu polymerase. 5 μl of the product from the DNA directed DNA synthesis was used as a template. The total reaction volume was 50 μl.
      Note: Initial denaturation step at 95 °C (5 min) followed by twenty-five PCR cycles at 94 °C (1 min), 50 °C (1 min), 72 °C (1 min), and finally a 5 min incubation at 72 °C were performed.
    4. The aqueous phase from the PCR reactions was phenol-chloroform extracted and recovered by ethanol precipitation as described above.
    5. PCR products were digested with 30 units each of EcoRI and PvuII in a total of 75 μl CutSmart buffer for 1 h at 37 °C.
    6. 15 μl of 6x gel loading dye was added to each reaction and samples were electrophoresed on a 12% non-denaturing polyacrylamide gel.
    7. The DNA was located by UV shadowing, recovered by overnight elution as described above, and spectrophotometry was used to quantify the recovered DNA.
      After preparing the inserts using the primer extension reactions and the PCR reactions described above, the plasmid vector was then prepared as described in DeStefano (2010). The inserts and the plasmid vector were ligated and the ligated plasmid was transformed into GC-5 E.coli cells as described in DeStefano (2010). Blue, faint blue, and white colonies were then counted and the colony mutation frequency of RT was determined using the number of each colonies.
      Note: All insertion or deletion frameshift mutations and most of the substitution mutations introduced by RT during either the RNA-directed or the DNA-directed synthesis steps will result in a faint blue or a white colony. The ratio of the number of faint blue/white colonies to the total number of colonies will give the colony mutation frequency (CMF). For example: In an experiment conducted at 6 mM Mg2+, 32 faint blue/white colonies were obtained along with 3605 blue colonies giving a CMF value of 8.8 x 10-3.

Recipes

  1. Extension reaction buffer (50 ml)
    1 M Tris HCl (pH 8)
    25 ml
    3 M KCl
    13.3 ml
    1 M DTT
    1ml
    RNase free water
    10.7 ml
  2. Elution buffer (50 ml)
    1 M Tris HCl (pH 7.5)
    500 μl
    0.5 M EDTA
    100 μl
    RNase free water
    49.4 ml
  3. 2x SDS loading buffer (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. DeStefano, J. J. (2010). Effect of reaction conditions and 3AB on the mutation rate of poliovirus RNA-dependent RNA polymerase in a alpha-complementation assay. Virus Res 147(1): 53-59.
  3. DeStefano, J., Ghosh, J., Prasad, B. and Raja, A. (1998). High fidelity of internal strand transfer catalyzed by human immunodeficiency virus reverse transcriptase. J Biol Chem 273(3): 1483-1489.
  4. 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.

简介

基于PCR的α-互补分析是测量聚合酶,特别是RNA依赖性RNA聚合酶(RDRP)和逆转录酶(RT)的保真度的有效技术。它已经成功地用于确定脊髓灰质炎病毒聚合酶3D-pol(DeStefano,2010)以及人类免疫缺陷病毒逆转录酶(HIV RT)的保真度(Achuthan等人,2014)。该测定法的主要优点是,由于涉及PCR步骤,甚至可以使用该测定法测量在两轮低合成RNA合成(对于RDRP)或逆转录(对于RT)后获得的产物的低产率。该测定也模拟逆转录过程,因为进行RNA和DNA指导的RT合成步骤。我们最近使用该测定法显示在生理相关镁浓度下的HIV RT具有与其它逆转录酶相同范围内的准确度(Achuthan等人,2014)。在这里,我们详细描述如何使用引物延伸反应准备插入。然后在基于PCR的α-互补分析中进一步处理制备的插入片段。

材料和试剂

  1. pBSM13 + (Stratagene)
  2. T3 RNA聚合酶(Roche Diagnostics,目录号:P2083)
  3. 10x转录缓冲液(Roche Diagnostics,目录号:P2083)
  4. 高保真PvuII(PvuII-HF)(New England Biolabs,目录号:R3151L)
  5. 高保真EcoRI(EcoRI-HF)(New England Biolabs,目录号:R3101L)
  6. 10x CutSmart缓冲液(New England Biolabs,目录号:R3101L)
  7. 6x凝胶上样染料(New England Biolabs,目录号:R3101L)
  8. NdeI(New England Biolabs,目录号:R0111L)
  9. T4多核苷酸激酶(PNK)(New England Biolabs,目录号:M0201L)
  10. 10×T4多核苷酸激酶缓冲液(New England Biolabs,目录号:B0201S)
  11. RNase(RNase抑制剂)(New England Biolabs,目录号:M0307L)
  12. RNase(无DNA酶)(Roche Diagnostics,目录号:11119915001)
  13. 无核糖核酸酶DNase I(Affymetrix,目录号:784111000)
  14. Pfu DNA聚合酶(Agilent Technologies,目录号:600353)
  15. 10x Pfu缓冲液(Agilent Technologies,目录号:600353)
  16. 核糖核苷三磷酸组(Roche Diagnostics,目录号:11277057001)
  17. 脱氧核苷三磷酸(dNTP)(Roche Diagnostics,目录号:11969064001)
  18. γ-[sup-32P] ATP(PerkinElmer,目录号:Blu502A001MC)
  19. G-25宏旋转柱(最适合体积为75-150μl)(哈佛仪器,目录号:74-3901)
  20. RNeasy RNA纯化试剂盒(QIAGEN,目录号:74104)
  21. 苯酚:氯仿:异戊醇(25:24:1)(Amresco,目录号:K169-400ML)
  22. 乙醇(VWR Lifesciences,目录号:EM1.00967.4003)
  23. 3M醋酸钠(Amresco,目录号:E521-100ML)
  24. 异丙醇(J.T.Baker ,目录号:9037-03)
  25. 40%丙烯酰胺 - 双丙烯酰胺(19:1)溶液(VWR International,目录号:JT4968-0)
  26. 40%丙烯酰胺 - 双丙烯酰胺(29:1)溶液(VWR International,目录号:JT4968-0)
  27. 尿素(VWR International,目录号:97061-926)
  28. 过硫酸铵(VWR International,目录号:97064-594)
  29. HIV逆转录酶[如Hou等人(2004)所述纯化]
  30. Milli-Q质量[RNase,无酶水(dH2O)]
  31. DNA寡核苷酸从Integrated DNA Technologies获得
  32. 延伸反应缓冲液(参见配方)
  33. 洗脱缓冲液(见配方)
  34. 2x SDS加样缓冲液(见配方)

设备

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

程序

  1. 底漆标签
    1. 所有引物应首先在50μl1×PNK缓冲液中放射性标记 连同50皮摩尔的每种引物,10μl[γ-32 P] ATP和5μl 单位的多核苷酸激酶(PNK)。 将反应混合物温育   在37℃温育30分钟,PNK在65℃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,并通过旋转除去过量的水 柱子在台式离心机以5000rpm离心4分钟
    3. 热灭活后,从中除去过量的[γ-32 P] ATP 反应混合物通过加载到平衡柱上并旋转 在5,000rpm下4分钟。

  2. 保真度测定的RNA的制备
    1. 得到用作保真度测定的模板的转录物 来自质粒pBSΔPvuII1146,其按照描述制备 DeStefano等人 (1998)。
    2. 对于RNA的制备,   质粒用50单位的酶NdeI在NEB缓冲液4中切割   在37℃下孵育3小时。 然后用苯酚提取裂解的质粒 氯仿萃取,乙醇沉淀回收
    3. 裂解后,跑出转录 在100μl的转录缓冲液中与2μg的转染缓冲液一起进行 线性化质粒,5μl100mM DTT,10μl5mM核糖核苷酸,   μl的RNA酶抑制剂和40单位的T3 RNA聚合酶在37℃下3小时 C。
    4. 向反应混合物中加入10单位DNA酶I 将反应物温育10分钟以消化剩余的DNA。
    5. 然后使用QIAGEN RNeasy试剂盒纯化RNA 制造商的说明书,并使用分光光度计量化。

  3. RNA指导的DNA合成
    1. 使用上述方法制备的约760nt的RNA模板   与放射性标记的25-nt DNA引物杂交 (5'-GCGGGCCTCTTCGCTATTACGCCAG-3')
    2. 对于杂交, 将引物加入25nM的模板(2:1比例的引物: 模板)在48μl的延伸反应缓冲液中以及6mM MgCl 2和100μMdNTP。 将混合物在65℃下加热5分钟 然后缓慢冷却至室温。 总反应体积为50   μl。
    3. 将引物 - 模板在37℃孵育3分钟。 2μl 的5μMHIV RT以引发延伸反应,
    4. RNA指导的DNA合成
      1. 使用上述方法制备的约760nt的RNA模板   与放射性标记的25-nt DNA引物杂交 (5'-GCGGGCCTCTTCGCTATTACGCCAG-3')
      2. 对于杂交, 将引物加入25nM的模板(2:1比例的引物: 模板)在48μl的延伸反应缓冲液中以及6mM MgCl 2和100μMdNTP。 将混合物在65℃下加热5分钟 然后缓慢冷却至室温。 总反应体积为50   μl。
      3. 将引物 - 模板在37℃孵育3分钟。 2μl 的5μMHIV RT以引发延伸反应,... 的合成。 来自DNA指导合成的产物看起来更暗 凝胶比第一轮的产品使用的是引物 此步骤具有较高的比活性
      4. 30分钟后,1 加入100μlRNase以消化剩余的RNA,样品为 加热至65℃5分钟以使RT失活
      5. DNA产物 然后通过标准苯酚氯仿萃取回收。 等于 体积(50μl)苯酚:氯仿:异戊醇混合物(25:24:1) 加入到反应中。 将反应物充分混合,然后旋入 微量离心机在18,000rpm离心3分钟。 水层从 移去顶部,转移到新管中并加入等体积(〜50μl)的 氯仿。 将混合物在18,000rpm下再次旋转3分钟   在微量离心机中,小心地除去水层 移动到一个新的管。
      6. 使用标准物沉淀DNA产物   乙醇沉淀。 加入两倍体积的100%乙醇(100μl) 加入十分之一体积(5μl)的3M乙酸钠并进行反应   在-20℃下贮存2小时
      7. 两个小时后, 反应以18,000rpm旋转20分钟。 上清液 仔细丢弃。 沉淀用500μl70%乙醇洗涤 然后干燥
      8. 将沉淀重悬于2x上样缓冲液中 并在6%变性7M上分离产物 脲 - 聚丙烯酰胺凝胶
      9. 从中切出199nt DNA产物   凝胶,将凝胶碎片粉碎并重悬于500μl洗脱液中 缓冲。 在4℃过夜洗脱后,将物质过滤 通过0.45μM注射过滤器,并使用回收DNA产物 苯酚氯仿萃取法。
      10. 的 将回收的产物重悬浮在洗脱缓冲液中,使用a 分光光度计,并用作DNA定向DNA的模板 合成步骤。

    5. DNA指导DNA合成
      1. 将从RNA定向合成回收的DNA产物杂交 到另一个20nt放射性标记的DNA引物 (5'-AGGATCCCCGGGTACCGAGC-3')具有至少10倍大的特异性 活性比用于第1轮的引物(见上文)。
      2. 一秒   如上所述进行一轮DNA合成,   反应体积为25μl。 保持延伸条件 在RNA和DNA模板延伸反应中相同。
      3. 用25μl2x加样缓冲液和产物终止反应 在8%变性7M尿素 - 聚丙烯酰胺凝胶上凝胶纯化。
        注意:凝胶运行足够远,可以有效地分离162-nt 从199-nt模板中完全扩展产品2(图2)。
      4. 定位162nt的产物并从凝胶切出。 产品   如上所述回收并重悬于100μl洗脱液中 缓冲。

    6. PCR反应
      1. 通过扩增在上述步骤中产生的第2轮DNA PCR使用以下引物:5'-GCGGGCCTCTTCGCTATTACGCCAG-3'和 5'-AGGATCCCCGGGTACCGAGC-3'。
      2. 第一个引物是用于的   RNA指导的DNA合成和第二引物与区域重叠 的原始RNA的质粒上的EcoRI位点 转录。
      3. 在购买的1x中进行PCR反应 Pfu缓冲液连同200μMdNTP,50皮摩尔每种引物,和5 单位的Pfu聚合酶。 5μl来自DNA定向DNA的产物 合成用作模板。 总反应体积为50μl 注意:初始变性步骤在95°C(5分钟),然后 在94℃(1分钟),50℃(1分钟),72℃(1分钟), 最后在72℃孵育5分钟。
      4. 的 来自PCR反应的水相是酚 - 氯仿萃取的   通过如上所述的乙醇沉淀回收
      5. PCR产物用EcoRI和PvuII各30单位在总共75μlCutSmart缓冲液中在37℃消化1小时。
      6. 向每个反应和样品中加入15μl6x凝胶负载染料 在12%非变性聚丙烯酰胺凝胶上电泳
      7. 通过UV遮蔽定位DNA,通过过夜洗脱回收 ,并使用分光光度法定量 回收DNA 在使用底漆制备插入物之后 延伸反应和PCR反应,质粒 载体如DeStefano(2010)所述制备。插入 连接质粒载体,连接质粒 转化到GC-5大肠杆菌细胞中,如DeStefano(2010)中所述。 然后计数蓝色,淡蓝色和白色菌落,并计数菌落 使用每个的数目确定RT的突变频率 群落。
        注意:所有插入或缺失移码突变和 大多数替代突变由RT引入 RNA指导或DNA指导的合成步骤将导致微弱 蓝色或白色殖民地。 淡蓝/白的数量比 菌落到总菌落数将给出菌落突变 频率(CMF)。 例如:在6mM Mg 2 + 进行的实验中, 连同3605个蓝色菌落一起获得32个淡蓝色/白色菌落   给出8.8×10 -3 的CMF值。

    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
      1ml
      无RNase水
      10.7 ml
    2. 洗脱缓冲液(50ml)
      1 M Tris HCl(pH 7.5)
      500微升
      0.5 M EDTA
      100微升
      无RNase水
      49.4毫升
    3. 2x SDS上样缓冲液(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体外生理镁条件下显示出更高的保真度。 J Virol 88(15):8514-8527。
    2. DeStefano,J.J。(2010)。 反应条件和3AB对脊髓灰质炎病毒RNA依赖性RNA聚合酶的突变率的影响, 互补测定。 Virus Res 147(1):53-59。
    3. DeStefano,J.,Ghosh,J.,Prasad,B.and Raja,A。(1998)。 人类免疫缺陷病毒逆转录酶催化的内部链转移的高保真度。 J Biol Chem 273(3):1483-1489。
    4. Hou,E.W.,Prasad,R.,Beard,W.A。和Wilson,S.H。(2004)。 高度表达和纯化未标记和组氨酸标记的HIV-1逆转录酶。 Protein Expr Purif 34(1):75-86。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Achuthan, V. and DeStefano, J. J. (2015). Primer Extension Reactions for the PCR- based α- complementation Assay. Bio-protocol 5(12): e1509. DOI: 10.21769/BioProtoc.1509.
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