欢迎您, 登录 | 注册

首页 | English

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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

系统生物学 > 基因组学 > 转座子
作者: Beery Yaakov
Beery YaakovAffiliation: Department of Life Sciences, Ben-Gurion University, Beer Sheva, Israel
Bio-protocol author page: a617
 and Khalil Kashkush
Khalil KashkushAffiliation: Department of Life Sciences, Ben-Gurion University, Beer Sheva, Israel
For correspondence: kashkush@bgu.ac.il
Bio-protocol author page: a399
Vol 3, Iss 11, 6/5/2013, 2866 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.788

[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.


How to cite this protocol: 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; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook