Stefan White, Michel Villerius, Martijn Breuning and Johan den Dunnen
Department of Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333AL Leiden, Nederland
The detection of copy number changes in complex genomes using DNA micro-arrays
(arrayCGH) is attracting much attention. Due to the complexity of a mammalian genome, the
technique is complicated and it is rather difficult to obtain a signal which is unique (no
background of repetitive DNA) and quantitative. Recently a method was published to detect
deletions/duplications in genomic DNA, designated Multiplex Amplifiable Probe
Hybridization (MAPH, Armour et al. [2000] Nucl.Acids Res. 28: 605-609). In this technique
a series of short DNA fragments are individually cloned in such a way that all can be
PCR-amplified using one pair of primers. These probes are hybridized to genomic DNA
immobilized on filters, and after stringent washing the probes are amplified off the
filters and used for seeding a second PCR. The products of this amplification are analyzed
on a polyacrylamide gel and the bands quantified using imaging apparatus.
We have applied MAPH to analyse DNA samples
from Duchenne/Becker Muscular Dystrophy (DMD/BMD) families. This disease is caused by
mutations of the dystrophin gene. In approximately 65% of cases the cause is found to be a
deletion or duplication of one or more of the 79 exons of the gene. Currently, multiplex
PCR on the most commonly affected exons is performed, but this allows the possibility of
missing smaller or rarer changes. Furthermore, Southern Blotting is required to detect the
exact borders of the deletions which determine whether the mutation causes a frame shift
(DMD) or not (BMD).
Each exon was cloned individually into the
pGEM-T-easy vector. The 79 probes were then divided into 2 pools and hybridized with the
filters. After the second PCR amplification using a fluorescently labeled primer the
products were analyzed on a capillary sequencer. The number of copies of each exon could
be determined by comparing the peak areas between controls and patients facilitating the
detection of 0:1, 1:2, 2:1 and 2:3 changes in copy number.
Work is currently being carried out regarding
automation of the technique, in both the hybridization and the subsequent data analysis.
Further probe sets are being developed to cover other regions of interest, and the
possibility of analyzing many more regions simultaneously using microarrays is being
investigated. We consider an array-based MAPH-approach as an attractive alternative
for genome-wide arrayCGH analysis.