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High Resolution Detection of Genetic Changes Associated with Transposons
高通量鉴定转座子及其侧翼序列-转座子展示联合454焦磷酸高通量测序法   

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Abstract

Transposable elements (TEs) are repetitive sequences, capable of inducing genetic mutations through their transpositional activity, or by non-homologous or illegitimate recombination. Because of their similarity and often high copy numbers, examining the effects of mutations caused by TEs in different samples (tissues, individuals, species, etc.) can be difficult. Thus, high throughput methods have been developed for genotyping TEs in un-sequenced genomes. A common method is termed Transposon Display (or transposon SSAP), which utilizes restriction enzymes and PCR amplification to produce chimeric DNA molecules that include genomic and TE DNA. The advent of second generation sequencing technologies, such as 454-pyrosequencing, have dramatically improved the resolution of this assay, allowing the simultaneous sequencing of all PCR products, representing all amplified TE sites in a specific genome.

Keywords: Transposon(转座子), PCR(PCR), Sequencing(测序), Restriction(限制), AFLP(AFLP)

Materials and Reagents

  1. Two oligonucleotides which form the double-stranded adapter, with an overhang complementary to the overhand of the restriction enzyme used. In the case of Mse I, the overhang is a 5' TA, and the adapter sequences are 5'- TACTCAGGACTCAT-3' and 5'- GACGATGAGTCCTGAG-3'. The two nucleotides at the 5' end of the former oligonucleotide will constitute the 5' TA overhang, after hybridization of the two sequences (green rectangles in Figure 1). 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 C nucleotide at the 3' end (5'-GATGAGTCCTGAGTAAC-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 (TTAA) between the primer and the 5' end of the TE should be avoided. Both the adapter- and TE-specific primers should be designed with the linker A and B sequences (for 454-pyrosequencing) at their 5' ends.
  3. NaCl
  4. T4 DNA ligase and buffer (New England Biolabs, catalog number: M0202 )
  5. Restriction enzyme MseI (New England Biolabs, catalog number: R0525 )
  6. Taq DNA polymerase and Taq DNA polymerase buffer (EURx, catalog number: E2500 )
  7. MgCl2
  8. dNTP mix


    Figure 1. An overview of the TD-454 pyrosequencing method. (a) The genomic samples are fragmented with a restriction enzyme and ligated to adapters (green rectangles); (b) The fragments undergo PCR with a primer specific to the adapter (containing a 454 linker sequence) and a primer specific to the analyzed TE (containing a 454 linker sequence); (c) The resulting PCR amplicons are sequenced with a 454 pyrosequencer.


Equipment

  1. Thermal cycler

Procedure


A.  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.


B.  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 Mse I, 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.


C. 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 adapter-specific pre-selective primer,1 μl of 50 ng/ul transposon-specific primer, 4 μl of Restriction/Ligation reaction products (cut with Mse I) 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 sec
    3. 60 °C for 30 sec
    4. 72 °C for 1 min
      Return to stage b for 29 times.
  3. Run the relevantly-sized PCR products (usually a narrow band between 150-550 bp, such as 300-500 bp, depending on the expected number of amplicons) in a 454-pyrosequencing machine (Figure 2).


    Figure 2. An example of PCR products from pre-selective amplification, using a primer from the adapter and a prom from a Stowaway-like MITE, called Eos, on a 1.5% agarose gel. The lanes include a 100 bp marker (left), pre-selective amplification of goatgrass Aegilops longissima (accession TL05; middle lane) and pre-selective amplification of diploid wheat Triticum urartu (accession TMU06; right lane).

Acknowledgments

The transposon (TD) display method was adapted from the amplified fragment length polymorphism (AFLP) (Vos et al., 1995 Nucl Acid Res 23:4407-4414). This work was supported by a grant from the Israel Science Foundation (grant # 142/08) to Khalil Kashkush.

References

  1. Yaakov, B. and Kashkush, K. (2012). Mobilization of Stowaway-like MITEs in newly formed allohexaploid wheat species. Plant Mol Biol 80(4-5): 419-427.

简介

转座元件(TE)是重复序列,能够通过其转座活性或通过非同源或非同源重组诱导遗传突变。 由于它们的相似性和通常高的拷贝数,检查由不同样品(组织,个体,物种等)中TE引起的突变的影响可能是困难的。 因此,已经开发了用于在未测序的基因组中对TE进行基因分型的高通量方法。 常见的方法称为转座子展示(或转座子SSAP),其利用限制酶和PCR扩增来产生包括基因组和TE DNA的嵌合DNA分子。 第二代测序技术(例如454焦磷酸测序)的出现已显着改善了该测定的分辨率,允许同时测序所有PCR产物,代表特定基因组中的所有扩增的TE位点。

关键字:转座子, PCR, 测序, 限制, AFLP

材料和试剂

  1. 形成双链衔接子的两个寡核苷酸,其具有与使用的限制酶的外切互补的突出端。在Mse I的情况下,突出端是5'TA,接头序列是5'-TACTCAGGACTCAT-3'和5'-GACGATGAGTCCTGAG-3'。在两个序列杂交后(图1中的绿色矩形),前寡核苷酸5'端的两个核苷酸将构成5'TA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C核苷酸(5'-GATGAGTCCTGAGTAAC-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(TTAA)。衔接头和TE特异性引物应当在其5'末端设计有接头A和B序列(用于454-焦磷酸测序)。
  3. NaCl
  4. T4 DNA连接酶和缓冲液(New England Biolabs,目录号:M0202)
  5. 限制酶MseI(New England Biolabs,目录号:R0525)
  6. Taq DNA聚合酶和Taq DNA聚合酶缓冲液(EURx,目录号:E2500)
  7. MgCl 2
  8. dNTP mix


    图1. TD-454焦磷酸测序方法的概述(a)基因组样品用限制酶片段化, 连接到适配器(绿色矩形); (b)片段经历PCR 与适配子特异性的引物(含有454接头序列)   和对分析的TE特异性的引物(含有454接头) 序列); (c)将所得PCR扩增子用454进行测序 焦磷酸测序仪。


设备

  1. 热循环仪

程序


A.  适配器对准备

  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


B.  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Mse I,300-500 ng的基因组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


C.预选择性扩增

  1. 加入0.2ml管中:2μl10x 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衔接子特异性预选择性引物,1μl50ng /μl转座子特异性引物,4μl限制性/连接反应产物(用Mse I I切割)和ddH 2 O,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℃30秒
    3. 60℃,30秒
    4. 72℃1分钟
      返回阶段b 29次。
  3. 在454-焦磷酸测序机中运行相关大小的PCR产物(通常在150-550bp之间的窄带,例如300-500bp,取决于预期的扩增子数)(图2)。

    图2。 使用来自衔接子的引物和来自类似Stowaway的MITE的启动子(称为 Eos )的预选择性扩增的PCR产物的实例, (登录号TL05;中间泳道)的预先选择性扩增,以及预选择性扩增的山羊草二倍体小麦(登录号TMU06;右泳道)。

致谢

转座子(TD)展示方法适应于扩增片段长度多态性(AFLP)(Vos等人,1995 Nucl Acid Res 23:4407-4414)。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. Yaakov,B。和Kashkush,K。(2012)。新近调用类似偷渡的MITE形成的异源六倍体小麦物种。 植物分子生物学 80(4- 5):419-427
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引用:Yaakov, B. and Kashkush, K. (2013). High Resolution Detection of Genetic Changes Associated with Transposons. Bio-protocol 3(11): e788. DOI: 10.21769/BioProtoc.788.
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