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A Quick, No Frills Approach to Mouse Genotyping
小鼠基因型快捷分析法   

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Abstract

Mice are extremely powerful mammalian genetic model organisms for basic and medical research, but managing a colony of transgenic mice is time consuming and expensive, many times requiring the help of dedicated technicians. Slow and laborious genotyping procedures add to the hassle. Outsourcing is costly and may not be as fast as desired, especially when setting up time sensitive experiments. Ultrafast genotyping protocols often require real-time PCR instruments and commercial reagents that may not be economical or practical. This protocol, adapted from methods suggested by The Jackson Laboratory, employs a minimalist approach that maximizes convenience by simplifying the tissue digestion/DNA extraction process and using a high-speed electrophoresis system for sample analysis. Genotype PCR results can be obtained in 3 h or less for as many samples as can fit in a PCR machine or can be efficiently handled by a user. Subsequent ethanol or chloroform purified DNA can be used in a standard PCR reaction to roughly identify a homozygous and a hemizygous mouse.

Materials and Reagents

  1. NaOH (NaOH pellets)
  2. Taq DNA Polymerase with ThermoPol buffer (New England Biolabs, catalog number:  M0267X *, M0267L , or M0267S )
    * Note: At 4,000U, ~800 μl of Taq serves several thousand PCR reactions. Buffer becomes a limiting reagent. ThermoPol Buffer recipe is available at NEB website. This buffer can be ordered separately from NEB (New England Biolabs, catalog number:  B9004S )
  3. Primers, recommend to be 18-21 bp in length, have a melting temperature above 56 °C and around 58 °C, and produce amplicons of 150-600 bp.
  4. DNA loading buffer
    Recommend Orange G (Sigma-Aldrich, catalog number:  O3756 ) instead of bromophenol blue for loading dye
  5. DNA ladder range for 100-800 bp range
    Recommend 1 kb Plus DNA ladder (Life Technologies, catolog number: 10787-018 )
  6. Agarose
  7. EtBr (Sigma-Aldrich, catalog number:  E8751 ) or SYBR Safe DNA Gel Stain (Life Technologies, catalog number: S33102 )
  8. NaOAc
  9. EtOH
  10. Phenol/Chloroform/Isoamyl alcohol (25: 24: 1) (Do not use acid phenol)
  11. 50 mM NaOH in dH2O (see Recipes)
  12. 10 mM dNTP Mix  (see Recipes)
  13. 1x TAE buffer (see Recipes)
  14. dNTP set 100 mM each A, C, G, T (GE Life Sciences, catalog number: 28406552 ) (see Recipes)
  15. 0.3 M NaOAc in ddH2O (see Recipes)

Equipment

  1. PCR Thermal cycler (96 well capacity preferred)
  2. Centrifuges (mini for PCR tubes and microcentrifuge for 1.5 ml tubes)
  3. Liberty 1 buffer-less high speed gel system (Neuvitro, 6Mgel - SYS-LBT1)
  4. Liberty 1, 12-channel pipette compatible 13 teeth combs (Neuvitro, 6Mgel - CMS-1315)
  5. Multichannel Pipette 2-20 μl
  6. Repeat Pipettor 10-125 μl
  7. PCR tube with cap, 8 or 12 PCR strip tubes, or 96-well PCR plate

Procedure

  1. Part I. Digest tissue for genotyping
    Note: This protocol is performed on 1-48 samples at the same time using 12-strip PCR tubes. The strip PCR tubes allow the use of multichannel pipettes to transfer solutions. 96 PCR plates can be used as well.
    1. Tissue digestion:
      1. Place a roughly 2-3 mm ear clip, tail, or other tissue biopsy in each PCR tube as they are obtained.
      2. Add 75 μl of 50 mM NaOH to PCR tube. Make sure tissue sample is submerged.
        Note: For adult mice ear clips add 75 μl, for adult tail clips 100 μl is preferred, and for neonatal mice tail or toe clips add 75 μl of 50 mM NaOH. Less than 75 μl makes it more difficult to use a repeat pipetor to squirt out a sufficient volume of 50 mM NaOH into each PCR tube.
      3. Incubate in thermo cycler for 95 °C for 30 min to 1 h. 45 min is recommended.
        Note: Samples have been heated for up to 2 h and as little as 15 min. 15-30 min is sufficient for neonatal tissue.
      4. Immediately flick tubes.
        Key feature: Tissue sample should partially break apart when PCR tube is flicked several times while still hot. Liquid should be cloudy. If tissue is not falling apart, incubate for longer.
      5. Allow samples to cool to room temperature.
      6. Briefly spin to remove liquid from caps.
      7. Proceed to Part II or store samples at room temperature overnight for next day use.
        Notes:
        1. Samples can be kept at room temperature for up to 3 days. Samples left for over a week have been used successfully but not recommended.
        2. Freeze at -20 °C for long-term storage. Neutralization step for base pH created by NaOH is not required but may benefit long-term storage. 10x ThermolPol buffer or 1 volume of 0.3 M NaOAc in dH2O can be used to neutralize.

  2. Part II. PCR rection and gel electrophoresis
    1. PCR after tissue digestion:
      1. PCR master mix setup.
        Note: Always make for more reactions than needed. For every 12 samples make for 2 samples extra, e.g. for 48 samples, make master mix for 56 reactions total.
        Per reaction:
        20 μl – dH2O
        2.5 μl – 10x ThermoPol buffer
        0.5 μl – 10 mM dNTPs
        0.5 μl – 10 μM primer mix
        0.1 μl – Taq DNA Polymerase
        23.6 μl total
      2. Aliquot 24 μl of master mix into new PCR tube(s) at room temperature.
      3. Transfer 2 μl of DNA from PCR tubes containing digested tissue (Part I) to PCR tubes containing aliquot of PCR master mix.
      4. Briefly spin.
      5. Samples can be kept at 4 °C until a PCR machine is available same day.
      6. PCR program
        1. 95 - 2 min
        2. 95 - 30 sec
        3. 56 - 30 sec
        4. 72 - 30 sec
        5. repeat steps ii, iii, iv 34x
        6. 72 - 1 min
        7. 10 - 5 min
          END – completed reactions can be left at room temperature until ready for gel electrophoresis.
          Tip: Do not modify the PCR reaction or program. Instead design all primers to be suitable under the same reaction condition. This way you can run multiple PCRs with different primers at the same time. This approach has been used to genotype mice containing multiple transgenes and for background backcrossing1,2
    2. 6 min gel electrophoresis.
      1. Cast a 2% agarose gel with EtBr or SYBR DNA gel stain in a liberty 1 apparatus (use 4 x 13 well combs to be able to load 48 samples and DNA ladder).
      2. Add 5 μl of DNA loading buffer to each PCR sample.
      3. Mix by pipetting up and down.
      4. Transfer 15 μl to 12 wells per lane in 2% agarose gel.
      5. Add DNA ladder with loading buffer to 13th well.
      6. Run gel electrophoresis at 200-220V for 6 min.
      7. Use appropriate gel viewer or imaging apparatus to identify bands.

  3. Part III. Purifying genomic DNA and subsequent use
    1. You can purify DNA from digest in Part I in various ways. Two are listed:
      1. Precipitating DNA (crude purification suitable for a primer sequencing reaction or semi-quantitative PCR).
        1. Allow debri to settle after digestion procedure in Part I.
        2. Transfer top 50 μl of solution into 1.5 ml tube(s). Avoid picking up debri.
        3. Add 50 μl of 0.3 M NaOAc in dH2O, mix by flicking tubes.
        4. Add 300 μl of 100% EtOH, mix by flicking tubes.
        5. Incubate in -80 °C for 3 min.
        6. Centrifuge at 16,000 x g for 3 min.
        7. Dry pellet and resuspend in 50 μl dH2O or preferred buffer.
        8. DNA concentration can now be assessed by spectrophotometry.
      2. Phenol/Chloroform DNA purification (stringent purification)
        1. Allow debri to settle after digestion procedure in Part I.
        2. Transfer 50 μl of solution into 1.5 ml tube(s). Avoid picking up debri.
        3. Add 150 μl of 0.3 M NaOAc in dH2O, mix by flicking tubes.
        4. Add 200 μl of Phenol/Chloroform/Isoamyl alcohol (25: 24: 1), shake tubes.
        5. Centrifuge at 16,000 x g for 5 min.
        6. Transfer 150 μl of top phase to new 1.5 ml tube.
        7. Add 450 μl of 100% EtOH.
        8. Place tubes in -80 °C for 3 min.
        9. Centrifuge at 16,000 x g for 3 min.
        10. Remove supernatant without disturbing pellet.
        11. Add 1 ml of 75% EtOH and shake tube.
        12. Centrifuge at 16,000 x g for 3 min.
        13. Dry pellet and resuspend in 50 μl dH2O or preferred buffer.
        14. DNA concentration can now be assessed by spectrophotometry.
    2. Purified DNA can be used to determine homozygote and heterozygote mice by semi-quantitative PCR
      Note: This method will increase the chances of identifying a mouse homozygous for a gene if selective primers are not available. This method was used to select homozygote parent mice for either a transgene containing the tetracycline transactivator or yellow fluorescent protein1.
      1. 2 μl of 0.05 μg μl-1 DNA can be used in PCR reaction outlined above with PCR program cycle number adjusted to 19-23x, 21x recommended.
        Tip: PCR cycle number should be optimized to when a positive band for a given primer set can just be seen for a known heterozygous sample.
      2. After gel run and imaging, differences in band intensity can be compared by eye or software, such as ImageJ.
      Note: If a mouse is homozygous for a gene, the PCR band will be brighter than a sample from a mouse that is heterozygous for the gene. Tip: PCR reaction using primers for native genes in the genome can be used as controls.

Recipes

  1. 50 mM NaOH in dH2O
    Caution: NaOH (lye) solution is caustic! Wear gloves when handling.
  2. 10 mM dNTP Mix
    Recommend making mix from dNTP Set 100 mM each A,C,G,T
  3. 10 μM Primer mix
  4. 1x TAE buffer
  5. 0.3 M NaOAc in ddH2O

Acknowledgments

This protocol was adapted from previous work (Lopez et al., 2011; Lopez et al., 2012).

References

  1. Lopez, M. E., Klein, A. D., Dimbil, U. J. and Scott, M. P. (2011). Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder. J Neurosci 31(12): 4367-4378.
  2. Lopez, M. E., Klein, A. D., Hong, J., Dimbil, U. J. and Scott, M. P. (2012). Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C. Hum Mol Genet 21(13): 2946-2960.

简介

该实验方案的中文版正在准备中...

材料和试剂

  1. NaOH(NaOH颗粒)
  2. 具有ThermoPol缓冲液的Taq DNA聚合酶(New England Biolabs,目录号:M0267X *,M0267L或M0267S)
    *注意:在4,000U时,〜800μl的Taq提供几千个PCR反应。 缓冲液成为限制性试剂。 ThermoPol缓冲液配方可在NEB网站获得。 该缓冲液可以与NEB(New England Biolabs,目录号:B9004S)分开订购,
  3. 引物长度推荐为18-21bp,具有高于56℃和约58℃的解链温度,并产生150-600bp的扩增子。
  4. DNA加载缓冲液
    推荐Orange G(Sigma-Aldrich,目录号:O3756)代替溴酚蓝用于加载染料
  5. DNA梯度范围为100-800 bp范围
    推荐1 kb Plus DNA ladder(Life Technologies,产品编号:10787-018)
  6. 琼脂糖
  7. EtBr(Sigma-Aldrich,目录号:E8751)或SYBR Safe DNA Gel Stain(Life Technologies,目录号:S33102)
  8. NaOAc
  9. EtOH
  10. 苯酚/氯仿/异戊醇(25:24:1)(不要使用酸性苯酚)
  11. 50mM NaOH在dH 2 O中(见配方)
  12. 10 mM dNTP Mix(见配方)
  13. 1x TAE缓冲区(请参阅配方)
  14. dNTP集合A,C,G,T各100mM(GE Life Sciences,目录号:28406552)(参见配方)
  15. 0.3M NaOAc的ddH 2 O(参见配方)

设备

  1. PCR热循环仪(96孔容量优选)
  2. 离心机(微型PCR管和微量离心机1.5ml管)
  3. Liberty 1无缓冲高速凝胶系统(Neuvitro,6Mgel-SYS-LBT1)
  4. Liberty 1,12通道移液器兼容13牙梳(Neuvitro,6Mgel - CMS-1315)
  5. 多通道移液器2-20μl
  6. 重复移液器10-125μl
  7. 带帽的PCR管,8或12个PCR条带管或96孔PCR板

程序

  1. 用于基因分型的摘取组织
    注意:此协议同时使用12条PCR管对1-48个样品进行。 条带PCR管允许使用多通道移液管来转移溶液。 也可以使用96个PCR板。
    1. 组织消化:
      1. 在每个PCR管中放置一个大约2-3毫米的耳夹,尾巴或其他组织活检样本。
      2. 向PCR管中加入75μl50 mM NaOH。确保组织样品浸没。
        注意:对于成年小鼠耳夹增加75微升,对于成年尾夹100微升是优选的,对于新生小鼠尾巴或脚趾夹添加75微升50毫米的NaOH。小于75μl使得使用重复移液器在每个PCR管中喷出足够体积的50mM NaOH更困难。
      3. 在热循环仪中孵育95°C 30分钟至1小时。建议使用45分钟。
        注意:样品已加热至多2小时,少至15分钟。 15-30分钟足以治疗新生儿组织。
      4. 立即轻弹管。
        主要特点:当PCR管在仍然热的时候轻拂几次时,组织样品应部分分离。液体应多云。如果组织没有分开,孵育更长时间
      5. 让样品冷却至室温。
      6. 短暂旋转以去除盖帽上的液体。
      7. 进行第二部分或在室温下储存样品过夜,以备第二天使用 注意:
        1. 样品可以在室温下保存最多3天。 已停用一周以上的样品已成功使用,但不推荐使用。
        2. 在-20°C冷冻长期储存。 由NaOH产生的碱性pH的中和步骤不是必需的,但是可以有益于长期储存。 可以使用dH 2 中的10x ThermolPol缓冲液或1体积的0.3M NaOAc中和。

  2. 第二部分。 PCR反应和凝胶电泳
    1. 组织消化后PCR:
      1. PCR主混合设置。
        注意:始终要比所需更多的反应。 对于每12个样品,使2个样品额外,例如。 对于48个样品,使主混合物共56个反应。
        每个反应:
        20μl·dH 2 O
        2.5μl - 10x ThermoPol缓冲液
        0.5μl - 10 mM dNTPs
        0.5μl-10μM引物混合物
        0.1μl - Taq DNA聚合酶
        总共23.6微升
      2. 在室温下将24μl主混合物等分到新的PCR管中。
      3. 从含有消化组织(Part I)的PCR管中转移2μlDNA到含有PCR主混合物等分试样的PCR管中。
      4. 短暂旋转。
      5. 样品可以保持在4℃,直到PCR机器可用当天。
      6. PCR程序
        1. 95 - 2分钟
        2. 95 - 30秒
        3. 56 - 30秒
        4. 72 - 30秒
        5. 重复步骤ii,iii,iv 34x
        6. 72 - 1分钟
        7. 10 - 5分钟
          END - 完全反应可以在室温下保存,直到准备进行凝胶电泳 提示:不要修改PCR反应或程序。 而是设计所有引物在相同的反应条件下是合适的。 这样,您可以同时使用不同的引物运行多个PCR。 这种方法已经用于对含有多个转基因和背景回交的小鼠进行基因分型 1,2
    2. 6分钟凝胶电泳
      1. 在自由1装置中使用EtBr或SYBR DNA凝胶染色法进行2%琼脂糖凝胶(使用4×13孔梳子以能够装载48个样品和DNA梯)。
      2. 向每个PCR样品中加入5μlDNA上样缓冲液。
      3. 通过上下吹吸混合。
      4. 在2%琼脂糖凝胶中转移15μl至12孔/泳道。
      5. 加入DNA梯子加载缓冲液到第13孔。
      6. 在200-220V下进行凝胶电泳6分钟。
      7. 使用适当的凝胶观察器或成像仪器来识别条带
  3. 第三部分。 纯化基因组DNA并随后使用
    1. 你可以通过各种方式净化DNA从第一部分的消化。 两个列出:
      1. 沉淀DNA(适用于引物测序反应或半定量PCR的粗纯化)。
        1. 允许debri在第一部分消化程序后结算。
        2. 将顶部50μl的溶液转移到1.5 ml管中。 避免拾起debri。
        3. 加入50μl的0.3M NaOAc在dH 2 O中,通过轻弹管混合。
        4. 加入300微升100%乙醇,通过轻弹管混合。
        5. 在-80℃下孵育3分钟。
        6. 以16,000×g离心3分钟。
        7. 干沉淀并重悬于50μldH 2 O或优选缓冲液中。
        8. DNA浓度现在可以通过分光光度法评估。
      2. 苯酚/氯仿DNA纯化(严格纯化)
        1. 允许debri在第一部分消化程序后结算。
        2. 将50μl溶液转移到1.5ml管中。 避免拾起debri。
        3. 加入150μl的0.3M NaOAc在dH 2 O中,通过轻弹管混合。
        4. 加入200微升苯酚/氯仿/异戊醇(25:24:1),摇动管。
        5. 以16,000×g离心5分钟。
        6. 转移150微升的上阶段到新的1.5毫升管。
        7. 加入450μl的100%EtOH。
        8. 将管置于-80℃3分钟。
        9. 以16,000×g离心3分钟。
        10. 除去上清液,不干扰沉淀。
        11. 加入1ml 75%EtOH并摇动管。
        12. 以16,000×g离心3分钟。
        13. 干沉淀并重悬于50μldH 2 O或优选缓冲液中。
        14. DNA浓度现在可以通过分光光度法评估。
    2. 纯化的DNA可用于通过半定量PCR测定纯合子和杂合子小鼠 注意:如果没有选择性引物,这种方法将增加鉴定基因的纯合小鼠的机会。该方法用于选择纯合子亲本小鼠用于含有四环素反式激活蛋白或黄色荧光蛋白的转基因。
      1. 2μl的0.05μgμl -1 DNA可用于上述PCR反应,PCR程序循环数调整为19-23x,推荐21倍。
        提示:PCR循环数应该优化为给定引物组的正带可以正确地看到已知的杂合样品。
      2. 在凝胶运行和成像之后,可以通过眼睛或软件(例如ImageJ)比较条带强度的差异。
      注意:如果小鼠对于基因是纯合的,则PCR带将比来自与基因杂合的小鼠的样品更亮。提示:使用用于基因组中天然基因的引物的PCR反应可以用作

食谱

  1. 50mM NaOH,在dH 2 O中 注意:NaOH(碱液)溶液是苛性碱! 处理时戴上手套。
  2. 10 mM dNTP Mix
    推荐从dNTP制备混合物设置100 mM每个A,C,G,T
  3. 10μM引物混合物
  4. 1x TAE缓冲区
  5. 0.3M NaOAc在ddH 2 O中

致谢

该协议改编自以前的工作(Lopez等人,2011; Lopez等人,2012年)。

参考文献

  1. Lopez,M.E.,Klein,A.D.,Dimbil,U.J.and Scott,M.P。(2011)。 解剖定义的基于神经元的神经退行性尼古丁选择C型疾病的救援 J Neurosci 31(12):4367-4378。
  2. Lopez,M.E.,Klein,A.D.,Hong,J.,Dimbil,U.J.and Scott,M.P。(2012)。 神经元和上皮细胞拯救解决了脂质储存障碍中的慢性全身炎症Niemann-Pick C。 a> Hum Mol Genet 21(13):2946-2960。
  • 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。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lopez, M. E. (2012). A Quick, No Frills Approach to Mouse Genotyping. Bio-protocol 2(15): e244. DOI: 10.21769/BioProtoc.244.
  2. Lopez, M. E., Klein, A. D., Dimbil, U. J. and Scott, M. P. (2011). Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder. J Neurosci 31(12): 4367-4378.
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Thanks for writing such an easy-to-understand artclie on this topic.
6/17/2012 12:24:13 PM Reply