These 2 Pages of Supplementary Data Are to Be Placed on the WEBSITE ONLY (5 Copies)

These 2 Pages of Supplementary Data Are to Be Placed on the WEBSITE ONLY (5 Copies)

<p>These 2 pages of Supplementary Data are to be placed on the WEBSITE ONLY (5 </p><p> copies)</p><p>Online Repeat Assay Protocol:</p><p>CL3N58B PCR amplification across the DM2 CCTG repeat was performed as previously described2. Genomic Southern analysis was done using DNA digested with either BsoBI or EcoRI. To improve detection of the DM2 expansion we developed a repeat assay </p><p>(RA) based on previously published methods for DM1 and SCA108, 9. The RA amplifies the genomic DM2 region containing the CCTG repeat expansion, using primers CL3N58-</p><p>D R (5’-GGCCTTATAACCATGCAAATG-3’), </p><p>JJP4CAGG(5’-TACGCATCCGAGTTTGAGACGCAGGCAGGCAGGCAGGCAGG-</p><p>3’), and JJP3(5’-TACGCATCCGAGTTTGAGACG-3’), followed by Southern analysis of the PCR products probed with CL3N58-E R, an internal probe that binds to a unique sequence upstream of the CCTG repeat tract. JJP4CAGG contains the repeat sequence with a 5’ hanging tail with negligible complementarity to any known human sequence. </p><p>The repeat portion of JJP4CAGG binds randomly at multiple sites within an expanded </p><p>CCTG tract, giving rise to PCR products of varying sizes that migrate as a smear. The </p><p>JJP3 sequence is the same as the hanging tail sequence in JJP4CAGG and only primes from PCR products generated by using the hanging tail from JJP4CAGG as their template, making the PCR reaction more robust. Optimal amplification was found using </p><p>25l PCR with 200 M dNTPs, 50 mM Tris pH 9.1, 14 mM (NH4)SO4, 2 mM MgCl2, 0.4</p><p>M each for primers JJP4CAGG, JJP3, and CL3N58-D R, 0.1% Tween-20, 10% DMSO, and 0.75 U ProofSprinter enzyme (Thermo Hybaid, Franklin, MA). The PCR conditions were as follows: 95C, 15’; 35 x (94C 30”, 51C 30”, 72C 2’); 72C 10’. The temperature change from 94oC to 51oC is ramped at 2oC/sec. PCR products were run on a 0.8% agarose gel (1h, 150 V, TAE buffer), transferred to Hybond N+ membrane </p><p>(Amersham, Piscataway, NJ), hybridized in Rapid-Hyb buffer (Amersham, Piscataway, </p><p>NJ) at 60oC for 16 hours with an CL3N58E-R (5’-TTGGACTTGGAATGAGTGAATG-</p><p>3’) end-labeled with 33P--dATP according to the manufacturer’s specifications (NEB, </p><p>Beverly, Massachusetts). After hybridization the membrane was washed at 45oC in </p><p>2xSSC and 0.1% SDS and exposed to X-ray film. Because the ProofSprinter enzyme was discontinued we now perform the RA with Proof Pro DNA polymerase (Continental </p><p>Lab Products, San Diego, CA), using the manufacturer’s buffer and an annealing temperature of 55oC. </p>

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