Leiden Muscular Dystrophy pages

DGGE analysis of the DMD gene

(modified last December 7, 2003)


Contents



Introduction


Denaturing Gradient Gel-Electrophoresis (DGGE) is one of the most sensitive electrophoretic techniques to scan for mutations in genes. Two papers have described the use of DGGE for the analysis of the DMD-gene;


Our DMD DGGE-work


A paper describing our work (Hofstra et al.) is currently in press. The work described in this paper was presented on the 1998 meeting of the "American Society of Human Genetics" (Oct. 27-31, Denver, USA) - Abstract. For additional information contact Dr. Johan T. den Dunnen (ddunnen@lumc.nl) or Robert W.M. Hofstra (r.w.m.hofstra@med.rug.nl).

DGGE-segments were designed based on the melting profile of the exons. To allow efficient mutation detection, 15 exons were split in two segments, yielding in total 95 DGGE-amplicons. For primer sequences see "Primers for DGGE analysis of the dystrophin gene".

PCR-conditions: a 30 ul reaction-volume containing 250 uM dNTP’s, 1x Supertaqbuffer, 12.5 pmol of each primer and 1.5 U Amplitaq (Perkin Elmer). 10x Supertaqbuffer (Sphero-Q) contains 100 mM Tris-HCl (pH9.0), 500 mM KCl, 0.1% gelatin, 15 mM MgCl2 and 1% Triton X-100.

NOTE: use 3.0 mM MgCl2 for exons 3A, 3B and 75B, 8.5 mM MgCl2 for exons 13B, 18A, 43A and 61A, 6 mM MgCl2 for exon 63

PCR-program:

NOTE: for exons 3A en 3B use 5 min 96C, 35 cycles of 30 sec 94C, 30 sec 48 C and 1 min 72C, followed by 5 min 72C and denaturation/renaturation using 10 min 96C and 15 min 48C

NOTE: when patient DNA (male) is analysed, an equivalent amount of control DNA is added to ensure heteroduplex formation and to increas the sensitivity

Gel electrophoresis:


Dp427c and Dp427m


Using DGGE, Tubiello et al. (1995) studied the Dp427m muscle promoter region/exon 1 of the DMD-gene in 40 non-deleted, unrelated Italian patients (29 DMD and 11 BMD). In addition, in four patients with mental retardation, the Dp427c brain promoter region/exon 1 was studied as well. No changes were deteced. See also Primer sequences.



| Top of page | LMDp homepage | Diagnostic techniques |
| Remarks / information | Copyright, liability |