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Detection of DNA Methylation Changes Surrounding Transposable Elements
转座子侧翼区域DNA甲基化水平的检测   

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Abstract

Transposable elements (TEs) are a major component of all genomes, thus the epigenetic mechanisms controlling their activity is an important field of study. Cytosine methylation is one of the factors regulating the transcription and transposition of TEs, alongside Histone modifications and small RNAs. Adapter PCR-based methods [such as Amplified Fragment Length Polymorphism (AFLP)] have been successfully used as high-throughput methods to genotype un-sequenced genomes. Here we use methylation-sensitive restriction enzymes, in combination with PCR on adaptor-ligated restriction fragments, to evaluate epigenetic changes in TEs between genomic DNA samples.

Keywords: DNA methylation(DNA甲基化), Transposable elements(转座因子), TMD(TMD), Transposon methylation display(转座子甲基化显示), Cytosine methylation(Cytosine甲基化)

Materials and Reagents

  1. Two oligonucleotides which form the double-stranded adapter, with an overhang complementary to the overhang of the restriction enzyme used. In the case of HpaII or MspI, the overhang is a 5' CG, and the adapter sequences are 5'-GATCATGAGTCCTGCT-3' and 5'-CGAGCAGGACTCATGA-3'. The two nucleotides at the 5' end of the latter oligonucleotide will constitute the 5' CG overhang, after hybridization of the two sequences (black rectangles in Figure 1, Shaked et al., 2001). These oligonucleotides should be designed such that they do not resemble know sequences in the examined species.
  2. Pre-selective primers, one complementary to the adapter with the addition of a G nucleotide at the 3' end (5'-ATCATGAGTCCTGCTCGG-3'; primer P2 in Figure 1), and the other complementary to the TE of interest (primer P1 in Figure 1). The TE-specific primer should be designed as a reverse-complement of the 5' end of the TE with a Tm=60 °C, between 30-50 bp into the TE (to allow for sequence validation in downstream assays). Restriction enzyme recognition sites (CCGG) between the primer and the 5' end of the TE should be avoided.
  3. Selective primers, one identical to the above TE-specific primer with the addition of a fluorescent tag (e.g. 6-FAM) or radioactive tag (end label with 32P), and one similar to the pre-selective primer complementary to the adapter with the addition of random nucleotides at the 3' end (e.g. 5'-CATGAGTCCTGCTCGGTCAG-3', includes an extra TCAG at the 3' end).
  4. NaCl
  5. T4 DNA ligase and buffer (New England Biolabs, catalog number: M0202 )
  6. Restriction enzymes HpaII and MspI (New England Biolabs, catalog number: R0171 and R0106 )
  7. Taq DNA polymerase and Taq DNA polymerase buffer (EURx, catalog number: E2500 )
  8. MgCl2
  9. dNTP mix
  10. Polynucleotide Kinase (PNK) enzyme and PNK buffer (New England Biolabs, catalog number: M0201 )
  11. Gamma-phosphate (32P)-labeled ATP (or fluorescently-labeled primers)
  12. GS-500 ROX-labeled size standard (for fluorescently-labeled products only) (Applied Biosystems)
  13. Hi-Di Formamide (for fluorescently-labeled products only) (Applied Biosystems, catalog number: 4311320 )

    Figure 1. An overview of the TMD method, adapted from (Yaakov and Kashkush, 2011). The method steps include: (a) Restriction of genomic DNA with HpaII (H) or MspI (M); (b) The first round of PCR amplification, using a primer from within the TE (P1) and a primer from the adapter (P2); (c) The second round of PCR amplification, using primer P1 from within the TE, labeled with a radioactive or fluorescent tag, and primer P2 with the addition of random nucleotides at the 3' end; and (d) Electrophoresis of the resulting PCR amplicons on a polyacrylamide gel (in the case of a radioactive tag) or in a capillary fluorescence detection machine (in the case of a fluorescent tag).

Equipment

  1. Thermal cycler
  2. Agarose gel electrophoresis machine
  3. 43 cm PAGE machine (Thermo Scientific Owl Aluminum-Backed Sequencer S3S, for radiolabeled products only)
  4. Capillary electrophoresis machine (such as the Applied Biosystems 3730xl DNA analyzer; for fluorescently-labeled products only)

Procedure

I. Adapter pair preparation

  1. Mix the two adapter oligonucleotides to a final concentration of 250 ng/μl.
  2. Incubate them at 95 °C for 5 min and then at room temperature for 10 min.

II. Restriction/Ligation

  1. Add to a 0.2 ml tube: 1 μl of 10x ligase buffer, 1 μl of 0.5 M NaCl, 1 μl of the adapter pair 120 units of T4 ligase, 2 units of HpaII or MspI, 300-500 ng of genomic DNA and ddH2O to a final volume of 10 μl.
  2. Mix well and incubate at 37 °C for 2-3 h.
  3. Dilute reaction 1: 10 by adding 90 μl of ddH2O.
  4. This reaction can be stored at -20 °C.

III. Pre-selective amplification

  1. Add to a 0.2 ml tube: 2 μl of 10x Taq DNA polymerase buffer, 2 μl of 25 mM MgCl2, 0.8 μl of dNTP mix, 1 unit of Taq DNA polymerase, 1 μl of 50 ng μl-1 adapter-specific pre-selective primer, 1 μl of 50 ng/μl transposon-specific primer, 4 μl of Restriction/Ligation reaction products (cut with HpaII or MspI) and ddH2O to a final volume of 20 μl.
  2. Use the thermal cycler to PCR with the following program:
    1. 94 °C for 3 min
    2. 94 °C for 30 s
    3. 60 °C for 30 s
    4. 72 °C for 1 min
      Return to step b 29 times
  3. Run 10 μl of the resulting products on a 1.5% agarose gel to validate amplification.
  4. Dilute the remaining 10 μl with 190 μl of ddH2O.
  5. This reaction can be stored at -20 °C.

IV. Radiolabeling

  1. For 20 reactions, add to a 0.2 ml tube: 6 μl of ddH2O, 6 μl of transposon-specific primer, 2 μl of 10x PNK buffer, 1 μl of PNK and 5 μl of radiolabeled ATP.
  2. Mix well and incubate at 37 °C for 1 h and then at 70 °C for 10 min.

V. Selective amplification

  1. Add to a 0.2 ml tube: 2 μl of 10x Taq DNA polymerase buffer, 2 μl of 25 mM MgCl2, 0.8 μl of dNTP mix, 1 unit of Taq DNA polymerase, 1 μl of 50 ng μl-1 adapter-specific selective primer, 1 μl of radiolabeled (or fluorescently labeled) transposon-specific primer, 3 μl of pre-selective amplification PCR products and ddH2O to a final volume of 20 μl.
  2. Use the thermal cycler to PCR with the following program:
    1. 94 °C for 2 min.
    2. 63 °C for 30 sec (decrease temperature by 1 °C every cycle until 56 °C).
    3. 72 °C for 1 min.
      Return to step b 32 times.
  3. Run the resulting products on a denaturing 5% polyacrylamide gel (for radio-labeled products, see Figure 2 for an example); or add 0.5 μl of GS-500 ROX-labeled size standard, 1-2.5 μl of PCR product (add less PCR product if fluorescence intensity is too high) and complete to 13 μl with formamide (for fluorescently-labeled products only).

    Figure 2. An example (Yaakov and Kashkush, 2011) of an autoradiogram showing TMD products for 6 DNA samples representing two parental plants (P1 and P2) and their allopolyploid offspring (S1-S4). The TE analyzed is a Stowaway-like miniature inverted repeat transposable element (MITE), called Thalos. Arrow (a) shows a change in methylation between S1 and S2, as only the MspI band is present in P1 and S1-S2, but both HpaII and MspI bands are present in S3-S4. The disappearence (b) or appearance (c) of bands can also be seen, which may arise as a result of complete methylation of the restriction site, or a mutation in the restriction or primer binding sites. The total percent methylation of all sites for a given sample analyzed can be calculated by dividing the number of polymorphic sites (those resenting bands for only one restriction enzyme) by the total number of sites (presenting bands for one and both restriction enzymes).

Acknowledgments

The transposon methylation display method (TMD) was adapted from the amplified fragment length polymorphism (AFLP) (Vos et al. 1995), and used first by Shaked et al. (2001). This work was supported by a grant from the Israel Science Foundation (grant # 142/08) to Khalil Kashkush.

References

  1. Kashkush, K. and Khasdan, V. (2007). Large-scale survey of cytosine methylation of retrotransposons and the impact of readout transcription from long terminal repeats on expression of adjacent rice genes. Genetics 177(4): 1975-1985.
  2. Shaked, H., Kashkush, K., Ozkan, H., Feldman, M. and Levy, A. A. (2001). Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat. Plant Cell 13(8): 1749-1759.
  3. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and et al. (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23(21): 4407-4414.
  4. Yaakov, B. and Kashkush, K. (2011) Massive alterations of the methylation patterns around DNA transposons in the first four generations of a newly formed wheat allohexaploid. Genome 54: 42-49.

简介

转座元件(TE)是所有基因组的主要组成部分,因此控制其活性的表观遗传机制是重要的研究领域。 胞嘧啶甲基化是调节TE的转录和转座,与组蛋白修饰和小RNA一起的因素之一。 基于适配子PCR的方法[例如扩增片段长度多态性(AFLP)]已经成功地用作高通量方法来对未测序的基因组进行基因分型。 在这里我们使用甲基化敏感的限制酶,结合适配器连接的限制性片段上的PCR,以评估基因组DNA样品之间的TE的表观遗传变化。

关键字:DNA甲基化, 转座因子, TMD, 转座子甲基化显示, Cytosine甲基化

材料和试剂

  1. 形成双链衔接子的两个寡核苷酸,其具有与所使用的限制酶的突出端互补的突出端。在HpaII或MspI的情况下,突出端是5'CG,接头序列是5'-GATCATGAGTCCTGCT-3'和5'-CGAGCAGGACTCATGA-3'。在两个序列杂交后(图1中的黑色矩形,Shaked等人,2001),在后者寡核苷酸的5'末端的两个核苷酸将构成5'CG突出端。这些寡核苷酸应该被设计为使得它们不类似于已知物种中的已知序列
  2. 预选择性引物,其与衔接子互补,在3'末端添加G核苷酸(5'-ATCATGAGTCCTGCTCGG-3';图1中的引物P2),并且另一个与感兴趣的TE互补(引物P1在图1)。 TE特异性引物应设计为TE的5'末端的反向互补,Tm = 60℃,在TE之间30-50bp之间(以允许在下游测定中的序列验证)。应避免引物和TE的5'端之间的限制酶识别位点(CCGG)
  3. 选择性引物,其与添加有荧光标签(例如6-FAM)或放射性标签(具有 32 P的末端标记)的上述TE特异性引物相同,并且一个类似于与衔接子互补的在3'末端添加随机核苷酸(例如,5'-CATGAGTCCTGCTCGGTCAG-3')的预选择引物在3'端包括额外的TCAG )。
  4. NaCl
  5. T4 DNA连接酶和缓冲液(New England Biolabs,目录号:M0202)
  6. 限制性酶HpaII 和 MspI(New England Biolabs,目录号:R0171和R0106)
  7. Taq DNA聚合酶和Taq DNA聚合酶缓冲液(EURx,目录号:E2500)
  8. MgCl 2
  9. dNTP mix
  10. 多核苷酸激酶(PNK)酶和PNK缓冲液(New England Biolabs,目录号:M0201)
  11. γ-磷酸(标记的32 P)标记的ATP(或荧光标记的引物)
  12. GS-500 ROX标记大小标准品(仅适用于荧光标记产品)(Applied Biosystems)
  13. Hi-Di甲酰胺(仅用于荧光标记的产物)(Applied Biosystems,目录号:4311320)

    图1. TMD方法的概述,改编自(Yaakov和Kashkush,2011)。  方法步骤包括:(a)用HpaII(H)或MspI(M)限制基因组DNA; (b)第一轮PCR扩增,使用引物 TE(P1)内的引物和来自衔接子(P2)的引物; (c)第二 使用来自TE内的标记的引物P1进行一轮PCR扩增 具有放射性或荧光标记,和具有添加的引物P2 的3'端的随机核苷酸;和(d) 得到的聚丙烯酰胺凝胶上的PCR扩增子(在a 放射性标签)或毛细管荧光检测机(in 荧光标签的情况)。

设备

  1. 热循环仪
  2. 琼脂糖凝胶电泳仪
  3. 43cm PAGE机(Thermo Scientific Owl Aluminum-Backed Sequencer S3S,仅用于放射性标记产物)
  4. 毛细管电泳仪(如Applied Biosystems 3730xl DNA分析仪;仅适用于荧光标记的产品)

程序

I.适配器对制备

  1. 混合两个适配器寡核苷酸至250 ng /μl的终浓度
  2. 在95℃孵育5分钟,然后在室温孵育10分钟

II。 限制/连接

  1. 加到0.2ml试管中:1μl10×连接酶缓冲液,1μl0.5M NaCl,1μl衔接子对120单位T4连接酶,2单位HpaII或MspI ,300-500ng基因组DNA和ddH 2 O至最终体积为10μl。
  2. 充分混合并在37℃孵育2-3小时
  3. 通过加入90μlddH 2 O稀释反应1:10
  4. 该反应可以在-20℃下保存

III。 预选择性扩增

  1. 加到0.2ml管中:2μl10×Taq DNA聚合酶缓冲液,2μl25mM MgCl 2,0.8μldNTP混合物,1单位Taq DNA聚合酶,1μl50ng /μl -1 衔接头特异性预选择引物,1μ μl的50ng /μl转座子特异性引物,4μl的限制性/连接反应产物(用HpaII或MspI切割)和ddH 2 - O至最终体积为20μl
  2. 使用热循环仪进行PCR,使用以下程序:
    1. 94℃3分钟
    2. 94°C 30秒
    3. 60°C 30秒
    4. 72℃1分钟
      返回步骤b 29次
  3. 在1.5%琼脂糖凝胶上运行10μl所得产物以验证扩增
  4. 用190μlddH 2 O稀释剩余的10μl
  5. 该反应可以在-20℃下保存

IV。 放射性标记

  1. 对于20个反应,加入0.2ml试管:6μlddH 2 O,6μl转座子特异性引物,2μl10×PNK缓冲液,1μlPNK和5μl放射性标记的ATP 。
  2. 充分混合并在37℃孵育1小时,然后在70℃孵育10分钟

V.选择性扩增

  1. 加到0.2ml管中:2μl10×Taq DNA聚合酶缓冲液,2μl25mM MgCl 2,0.8μldNTP混合物,1单位Taq DNA聚合酶,1μl50ng /μl 接头特异性选择性引物,1μl放射性标记(或荧光标记的)转座子特异性引物,3μl预选择性扩增PCR产物和ddH 2 O至 最终体积为20μl
  2. 使用热循环仪进行PCR,使用以下程序:
    1. 94℃2分钟
    2. 63℃30秒(每个循环降低温度1℃,直到56℃)
    3. 72℃1分钟。
      返回步骤b 32次。
  3. 在变性5%聚丙烯酰胺凝胶(用于放射性标记产物,参见图2的实例)上运行所得产物;或加入0.5μlGS-500 ROX标记的大小标准物,1-2.5μlPCR产物(如果荧光强度太高,则添加较少的PCR产物),并用甲酰胺(仅用于荧光标记的产物)完成至13μl。 br />
    图2.显示表示两个亲本植物(P1和P2)及其异源多倍体后代(S1-S4)的6个DNA样品的TMD产物的放射自显影图的实例(Yaakov和Kashkush,2011)。 被分析的TE是一种 Stowaway 样微型反转重复转座元件(MITE),称为 Thalos 。箭头(a)显示了S1和S2之间的甲基化的变化,因为在P1和S1-S2中仅存在MspI 条带,而在mspI和em- 频段存在于S3-S4中。还可以看到条带的消失(b)或外观(c),这可能是限制性位点的完全甲基化或限制性或引物结合位点中的突变的结果。对于分析的给定样品,所有位点的总百分比甲基化可以通过将多态位点的数目(仅一种限制酶的那些重复条带)除以位点的总数(一种和两种限制性酶的呈现条带)来计算。 br />

致谢

转座子甲基化展示方法(TMD)从扩增的片段长度多态性(AFLP)(Vos等人,1995)改编,并且首先由Shaked等人使用( 2001)。这项工作得到了以色列科学基金会(拨款#142/08)给Khalil Kashkush的资助。

参考文献

  1. Kashkush,K。和Khasdan,V。(2007)。 反转录转座子的胞嘧啶甲基化的大规模调查和从长末端重复读出的转录对表达的影响 177(4):1975-1985 。
  2. Shaked,H.,Kashkush,K.,Ozkan,H.,Feldman,M。和Levy,A.A。(2001)。 序列消除和胞嘧啶甲基化是基因组对小杂交和异源多倍体的快速和可重复的反应。 植物细胞 13(8):1749-1759。
  3. Vos,P.,Hogers,R.,Bleeker,M.,Reijans,M.,van de Lee,T.,Hornes,M.,Frijters,A.,Pot,J.,Peleman,J.,Kuiper,M 。和等。 (1995)。 AFLP:一种新的DNA指纹识别技术。 Nucleic Acids Res < em> 23(21):4407-4414。
  4. Yaakov,B。和Kashkush,K。(2011) DNA转座子周围甲基化模式的大规模改变 54:42-49 。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yaakov, B. and Kashkush, K. (2013). Detection of DNA Methylation Changes Surrounding Transposable Elements. Bio-protocol 3(11): e784. DOI: 10.21769/BioProtoc.784.
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