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DGGE analysis of the DMD gene

(modified last December 7, 2003)

Contents

• Introduction
• All DMD exons
  • primers for DGGE-analysis
  • co-electrophoresis of exon products
• Dp427c and Dp427m

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;

• All DMD exons
  Hofstra RMW, Mulder IM, Vossen RHAM, de Koning-Gans PAM, Kraak M, Ginjaar HB, van der Hout AH, Bakker E, Buys CHCM, van Ommen GJB, van Essen AJ, den Dunnen JT (2003). Whole gene DGGE-based mutation scanning of the dystrophin gene in DMD/BMD patients. Human Mutation, in press.
• Exons Dp427c and Dp427m
  Tubiello G, Carrera P, Soriani N, Morandi L, Ferrari M. (1995). Mutational analysis of muscle and brain specific promoter regions of dystrophin gene in DMD/BMD Italian patients by denaturing gradient gel electrophoresis (DGGE). Mol.Cell Probes 9: 441-446.  -  Primer sequences

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:

• denaturation 5 min 96°C
• 5 cycles 30 sec 94°C, 30 sec 53°C (annealing T) and 1 min 72°C
• 5 cycles at annealing T 50°C
• 3 cycles at annealing T 48°C
• 15 cycles at annealing T 50°C
• 13 cycles at annealing T 48°C
• 5 min 72°C followed by denaturation/renaturation using 10 min 96°C and 15 min 48°C

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

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:

• gel:  9% gel (or 9-12%) with 15-55%, 20-60% or 35-75% ureum/formamide gradient (see below)
• electrophoresis:  16 h in 0.5x TAE-buffer at 58^oC
• exon products which we co-electrophorese
  • 15-55% UF gradient
    • 2, 1, 6A, 6B
    • 28, 33, 27, 35
    • 46, 32, 47
    • 44, 13A, 70
    • 14
  • 20-60% UF gradient
    • 12, 18B, 13B, 19
    • 34, 31, 49, 45
    • 53A, 58, 72
    • 66, 74, 77, 78
    • 73, 54B, 79, 67
    • 43A, 29A, 26A
    • 15, 29B, 30
    • 48, 16, 63
    • 20B, 43B, 50
    • 38, 36, 65
    • 5, 23A, 69
    • 39, 18A, 7
    • 68B, 53B, 55
    • 11, 25, 22
    • 10, 24, 51B
    • 8, 17, 4
    • 21, 9, 3B
    • 42, 64, 57
    • 20A, 51A, 76
    • 40, 52
    • 26B
    • Pm427m, Pm427c (exon 1)
  • 35-75% UF gradient
    • 23B, 37, 68A
    • 61B, 41A, 61A
    • 62, 41B, 54A
    • 56, 71, 60
    • 3A, 75B
    • 75A

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.

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