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