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Isolation of Genomic DNA from Mycobacterium Species
从分枝杆菌物种中分离基因组DNA   

Pawan KumarPawan Kumar*Soumitra MaratheSoumitra Marathe*Sangeeta BhaskarSangeeta Bhaskar  (*contributed equally to this work)
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Abstract

Isolation of genomic DNA from Mycobacterium species has been a tedious procedure. This can primarily be attributed to thick and waxy cell wall of mycobacteria which hampers lysis of the bacterial cell. We have tested various approaches to isolate mycobacterial DNA and based on this, an optimized protocol is presented here. This protocol involves initial incubation of mycobacteria with lysozyme, followed by SDS-proteinase K treatment to bring about cell disruption. In the case of slowly growing mycobacteria such as BCG (Bacillus Calmette–Guérin) or Mycobacterium tuberculosis (M. tuberculosis), an intermediate step of cell lysis by physical method results in significantly enhanced yield.

Materials and Reagents

  1. Mycobacterium culture (50 to 100 ml)
  2. Tris base (Sigma-Aldrich, catalog number: T1503 )
  3. EDTA (HiMedia Laboratories, catalog number: MB011 )
    Note: Prepare 0.5 M solution (pH 8) in double distilled water.
  4. Liquid nitrogen
  5. Proteinase K (HiMedia Laboratories, catalog number: RM2957 )
    Note: Prepare the stock solution of 10 mg/ml in TE buffer (pH 8).
  6. SDS (Bio Basic Canada, catalog number: SB0485 )
    Note: Prepare 10% solution by dissolving in double distilled water.
  7. Lysozyme (Sigma-Aldrich, catalog number: L6876 )
    Note: Prepare the stock solution of 100 mg/ml in double distilled water.
  8. Phenol (Sigma-Aldrich, catalog number: P4557 )
    Note: Equilibrate with Tris-Hcl (pH 8).
  9. Chloroform (HiMedia Laboratories, catalog number: AS041 )
  10. Isoamyl alcohol (HiMedia Laboratories, catalog number: MB091 )
  11. 2-Propanol (HiMedia Laboratories, catalog number: MB063 )
  12. Ethyl alcohol, Pure (Sigma-Aldrich, catalog number: 459844 )
  13. Nuclease-free water (HiMedia Laboratories, catalog number: TCL016 )
  14. TE buffer (see Recipes)
  15. PCI mixture (see Recipes)

Equipment

  1. Water bath
  2. Centrifuge
  3. Pestle and mortar

Procedure

  1. Harvest mycobacteria by centrifuging 20-40 ml log phase culture (O.D.600 ≈ 0.8) at 2,000 x g for 10 min, room temperature. Carefully discard culture medium and add 10 ml TE (at room temperature) buffer to pellet. Mix gently with a pipette.
    Note: If isolating DNA from pathogenic mycobacteria such as M. tuberculosis, step 1 and 2 must be performed in a BSL-3 facility.
  2. Incubate mycobacterial suspension at 80 °C for 1 h in a water bath. This step will facilitate loosening of the mycobacterial cell wall and will also inactivate pathogenic mycobacteria.
  3. Bring down temperature of suspension to ~30 °C and add 20 µl of 100 mg/ml lysozyme solution (final concentration, 2 mg/ml) to it. Incubate suspension at 37 °C for 6 h to overnight in a water bath.
    Note: In case of fast-growing mycobacteria such as Mycobacterium smegmatis (M. smegmatis), go directly to step 7. However, if DNA is to be isolated from slow-growing species, follow steps 4-6 which involve lysis of cells by a physical method. Because of enhanced disruption of cells, this would significantly enhance yield.
  4. Pellet down bacilli by centrifugation at 2,000 x g for 10 min, room temperature and carefully discard supernatant.
  5. Snap freeze pellet in liquid N2 and immediately transfer to a mortar. Using a pestle, crush bacteria while repeatedly adding liquid N2 (~20 ml) to it.
  6. Add 10 ml TE buffer into the mortar and carefully bring mycobacteria in suspension. Transfer this suspension to a 50 ml polypropylene tube.
  7. Add 1 ml of 10% SDS solution (final concentration, ~1%) and 20 µl of 10 mg/ml Proteinase K solution (final concentration 20 µg/ml). Incubate for 2 to 4 h at 60 °C in a hot water bath.
  8. Add an equal volume of PCI mixture to the tube and mix contents by gently inverting tube for 5-10 min.
  9. Centrifuge suspension at 12,000 x g for 10 min, 4 °C. Carefully transfer upper aqueous phase to a new 50 ml tube.
  10. Repeat steps 6 to 8 for achieving a higher purity of DNA.
  11. Add 100 µl 3 M sodium acetate solution per ml of aqueous phase obtained in step 10. Mix gently by inverting tube.
  12. Add an equal volume of isopropanol to tube. Mix contents gently and keep undisturbed for 5-10 min. The white thread-like structure of precipitated DNA will be seen in suspension (Figure 1).
  13. Centrifuge tube at 12,000 x g for 10 min, 4 °C. Discard liquid carefully.
  14. Wash DNA pellet by adding 10 ml of 75% ethanol. Centrifuge at 12,000 x g for 10 min, 4 °C.
  15. Aspirate liquid without disturbing pellet. Keep tube open to air dry pellet. Do not over dry pellet, as over dried DNA will be difficult to dissolve.
  16. Dissolve pellet in molecular biology grade water and analyze DNA by agarose gel electrophoresis and spectrophotometer for its purity and integrity.


    Figure 1. Precipitated mycobacterial DNA (as seen after step 12)

Recipes

  1. TE buffer
    50 mM Tris
    5 mM EDTA
    Use HCl to adjust pH to 8
  2. PCI mixture
    Tris-saturate phenol to pH 8
    Mix Phenol:Chloroform:Isoamyl alcohol in ratio of 25:24:1 volume by volume

References

  1. Amaro, A., Duarte, E., Amado, A., Ferronha, H. and Botelho, A. (2008). Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium. Lett Appl Microbiol 47(1): 8-11.
  2. Kumar, P., John, V., Marathe, S., Das, G. and Bhaskar, S. (2015). Mycobacterium indicus pranii induces dendritic cell activation, survival, and Th1/Th17 polarization potential in a TLR-dependent manner. J Leukoc Biol 97(3): 511-520.

简介

从分枝杆菌物种分离基因组DNA是一个冗长的过程。 这主要归因于分枝杆菌的厚的和蜡状的细胞壁,其阻碍细菌细胞的裂解。 我们已经测试了各种方法来分离分枝杆菌DNA,并基于此,这里介绍了一个优化的协议。 该方案包括分枝杆菌与溶菌酶的初始孵育,随后是SDS-蛋白酶K处理以引起细胞破裂。 在缓慢生长分枝杆菌如BCG(卡介苗)或结核分枝杆菌(<结核杆菌)的情况下,通过物理方法的细胞裂解的中间步骤导致 显着增加产量。

材料和试剂

  1. 分枝杆菌培养物(50至100ml)
  2. Tris碱(Sigma-Aldrich,目录号:T1503)
  3. EDTA(HiMedia Laboratories,目录号:MB011)
    注意:在双蒸水中制备0.5 M溶液(pH 8)。
  4. 液氮
  5. 蛋白酶K(HiMedia Laboratories,目录号:RM2957) 注意:在TE缓冲液(pH 8)中制备10 mg/ml的储备溶液。
  6. SDS(??Bio Basic Canada,目录号:SB0485)
    注意:溶解于双蒸水中制备10%的溶液。
  7. 溶菌酶(Sigma-Aldrich,目录号:L6876) 注意:在双蒸水中制备100 mg/ml的储备溶液。
  8. 苯酚(Sigma-Aldrich,目录号:P4557) 注意:用Tris-HCl(pH 8)平衡。
  9. 氯仿(HiMedia Laboratories,目录号:AS041)
  10. 异戊醇(HiMedia Laboratories,目录号:MB091)
  11. 2-丙醇(HiMedia Laboratories,目录号:MB063)
  12. 乙醇,Pure(Sigma-Aldrich,目录号:459844)
  13. 无核酸酶水(HiMedia Laboratories,目录号:TCL016)
  14. TE缓冲区(参见配方)
  15. PCI混合(见配方)

设备

  1. 水浴
  2. 离心机
  3. 杵和臼

程序

  1. 通过在2,000×g离心20-40ml对数期培养物(O.D. 600≈0.8)10分钟,室温下收获分枝杆菌。小心丢弃培养基并加入10ml TE(室温)缓冲液沉淀。用移液管轻轻混匀。
    注意:如果从致病性分枝杆菌如结核分枝杆菌中分离DNA,则必须在BSL-3设施中进行步骤1和2。
  2. 孵育分枝杆菌悬浮液在80℃水浴1小时。该步骤将有利于分枝杆菌细胞壁的松动,并且还将灭活病原分枝杆菌。
  3. 将悬浮液的温度降至?30℃,并向其中加入20μl的100mg/ml溶菌酶溶液(终浓度为2mg/ml)。在37℃下在水浴中孵育悬浮液6小时至过夜 注意:在快速生长的分枝杆菌如耻垢分枝杆菌(耻垢分枝杆菌)的情况下,直接进行步骤7.然而,如果要从缓慢生长的物种中分离DNA,则遵循涉及裂解的步骤4-6的细胞。由于细胞的增强的破坏,这将显着提高产量。
  4. 通过在2,000xg下离心10分钟,室温下并且仔细丢弃上清液,使下部杆菌沉淀。
  5. 在液氮中快速冷冻沉淀物并立即转移到研钵中。使用杵,粉碎细菌,同时重复地向其中加入液体N 2(?20ml)。
  6. 加入10ml TE缓冲液到砂浆中,小心使分枝杆菌悬浮。将此悬浮液转移到50ml聚丙烯管中
  7. 加入1ml的10%SDS溶液(终浓度?1%)和20μl的10mg/ml蛋白酶K溶液(终浓度20μg/ml)。在60°C的热水浴中孵育2至4小时
  8. 向管中加入等体积的PCI混合物,通过轻轻颠倒管混合内容物5-10分钟
  9. 在4℃下以12,000xg离心悬浮液10分钟。小心地将上层水相转移到新的50ml管中
  10. 重复步骤6到8以获得更高纯度的DNA
  11. 在步骤10中得到的每ml水相中加入100μl3M乙酸钠溶液。通过倒置管轻轻混合。
  12. 向管中加入等体积的异丙醇。轻轻混合内容,保持不受干扰5-10分钟。沉淀的DNA的白色线状结构将在悬浮液中看到(图1)
  13. 离心管在12,000×g下10分钟,4℃。小心丢弃液体。
  14. 通过加入10ml的75%乙醇洗涤DNA沉淀。在4℃下以12,000xg离心10分钟。
  15. 吸出液体,不要打扰沉淀。保持管开放至空气干燥颗粒。不要过度干燥颗粒,因为过干的DNA将难以溶解。
  16. 将颗粒溶解在分子生物学级水中,通过琼脂糖凝胶电泳和分光光度计分析DNA的纯度和完整性。


    图1.沉淀的分枝杆菌DNA(如步骤12后所示)

食谱

  1. TE缓冲区
    50 mM Tris
    5 mM EDTA
    使用HCl将pH调节至8
  2. PCI混合物
    Tris-饱和苯酚至pH 8
    混合苯酚:氯仿:异戊醇,体积比为25:24:1,体积比为

参考文献

  1. Amaro,A.,Duarte,E.,Amado,A.,Ferronha,H。和Botelho,A。(2008)。 比较牛分枝杆菌,分枝杆菌的三种DNA提取方法肺结核和分枝杆菌 avium subsp。 avium。 Lett Appl Microbiol 47(1):8-11。
  2. Kumar,P.,John,V.,Marathe,S.,Das,G.and Bhaskar,S。(2015)。 Mycobacterium indicus pranii 诱导树突状细胞活化,存活和Th1/Th17极化电位以TLR依赖性方式。 Leukoc Biol 97(3):511-520。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kumar, P., Marathe, S. and Bhaskar, S. (2016). Isolation of Genomic DNA from Mycobacterium Species. Bio-protocol 6(5): e1751. DOI: 10.21769/BioProtoc.1751.
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Gugu Musa
Durham University
I have 2 questions please
1. What are the appropriate conditions to lyse Mycobacterium smegmatis using sonication method (time, temperature, amplitude and others...)
2. Do I have to use Urea as part of my lysis buffer? If yes, at what concentration?
5/10/2016 7:01:31 AM Reply