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Rapid detection of genomic,,,duplications and deletions using,,,the Agilent 2100 bioanalyzer

but has proved technically difficult in practice. The fact that each primer pair has to be optimized when combined in a given PCR reaction limits the mix and match potential. FISH is a visual method, meaning that the number of signals seen corresponds to the copy number of the probe. The disadvantages are the time-consuming preprocessing necessary before the analysis can be performed and the relatively low resolution (approx. 40 kb for a cosmid probe). Although this is more than adequate for larger rearrangements, small (e.g. single exon) deletions and duplications are not usually detected with this method. Two high resolution, high throughput techniques have recently been described for quantitative analysis of DNA, namely multiplex amplifiable probe hybridization (MAPH)1 and multiplex ligation-dependent probe amplification (MLPA)2. Both are based on quantitative amplification using a single pair of primers following probe hybridization to genomic DNA. The difference lies in the probes and the way the genomic DNA is handled. MAPH probes are PCR products that are hybridized to genomic DNA immobilized onto a nylon membrane. After hybridization the unbound probes are removed by stringent washing, with the bound probes being subsequently denatured and amplified in a PCR reaction. As the probes are designed to all have identical sequences at each end, the PCR reaction can be performed with a single primer pair. MLPA is a ligation-based assay, with 2 single stranded DNA sequences being ligated together only if they both hybridize adjacently to their complementary sequences on genomic DNA. As unligated probes can not be PCR amplified it is not necessary to immobilize the genomic DNA (in contrast to MAPH), and the washing steps can also be skipped.
Different methods for the analysis and the quantitation of the PCR products have been describe
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