Materials and Reagents

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.
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.
NaCl
T4 DNA ligase and buffer (New England Biolabs, catalog number: M0202 )
Restriction enzyme MseI (New England Biolabs, catalog number: R0525 )
Taq DNA polymerase and Taq DNA polymerase buffer (EURx, catalog number: E2500 )
MgCl₂
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

Thermal cycler

Procedure

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.
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.
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

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.