Supporting information for Jansen et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.152330099

Technical Details

Total DNA was isolated from six hair-roots using 200 µl of lysis buffer (100 mM NaCl/25 mM EDTA) and 1 µl of proteinase K solution (20 mg/ml) at an incubation temperature of 60°C for 3-12 h. Purification was performed with the QIAamp Blood Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The control region was amplified using a semi-nested asymmetric PCR strategy. For the first PCR we used the published primers P1 and P2 (1). For the second PCR we designed a third primer, M13-HMT: 5'-TGT AAA ACG ACG GCC AGT ACC ATC AAC ACC CAA AGC-3'. The first 18 nucleotides are a sequencing tag (-21M13), and the remaining primer corresponds to nucleotide positions 15,425-15,442. The first PCR was performed with 1-5 µl of purified total DNA (20–200 ng), 50 µM of each primer, 0.2 µM dNTPs (Amersham Pharmacia), 5 µl 10´ buffer (Biomaster, Cologne, Germany), 1.5 mM MgCl2, and 1 unit bioTaq (Biomaster) in a total volume of 50 µl. Thermocycling was performed on a Biometra Trio-Thermoblock (Biometra GmbH, Göttingen, Germany) as follows: initial denaturing at 94°C for 5 min followed by 32 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, final extension at 72°C for 5 min. The 469-bp PCR product (nucleotide positions 15,395-15,862) was added without further purification to a second PCR, which was conducted just like the first PCR, except for the following components: 1-5 µl of the first PCR product (50-200 ng) as a template, 5 µM forward primer M13-HMT, and 0.0125 mM dNTPs. Thermocycling consisted in initial denaturing at 94°C for 3 min, then 32 cycles of 94°C for 30 s, 50°C for 50 s and 72°C for 2 min, and a final extension at 72°C for 5 min. The 437-bp PCR-product (nucleotide positions 15,425-15,862) was purified with the QIAquick PCR Purification kit (Qiagen) according to the manufacturer's instructions.

DNA sequencing was performed with the Dye Primer Cycle Sequencing Ready Reaction -21-M13 kit (Applied Biosystems, Weiterstadt, Germany) following the supplied protocol. Sequencing products were separated on an ABI 377 DNA Sequencer. The obtained sequences were aligned with the Sequence Editor Software version 1.0.3 (Applied Biosystems). The sequences were determined from nucleotide position 15,428 to nucleotide position 15,776 or up to nucleotide position 15,853.

1. Ishida, N., Hasegawa, T., Takeda, K., Sakagami, M., Onishi, A., Inumaru, S., Komatsu, M. & Mukoyama, H. (1994) Anim. Genet. 25, 215–221.