MLPA and MAPH: New techniques for detection of gene deletions

Abstract

For the Mutation Detection 2003 Special IssueScreening for deletions of all or part of genes poses a challenge in the diagnostic laboratory. Numerous methods are available for detecting deletions of a few base pairs or very large deletions, but difficulties arise in detecting deletions of a few kilobases. Two new techniques have recently been described that allow detection of such midsize deletions by simultaneously screening for the loss or duplication of up to 40 target sequences. These are the multiplex amplification and probe hybridization (MAPH) and the multiplex ligation-dependent probe amplification (MLPA). Both rely on sequence-specific probe hybridization to genomic DNA, followed by amplification of the hybridized probe, and semi-quantitative analysis of the resulting PCR products. The relative peak heights or band intensities from each target indicate their initial concentration. The two techniques differ in the ease with which probes can be generated in house, and the labor intensity of performing the assay.

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

“…Moreover, Southern blotting requires large amounts of high molecular weight DNA, and its interpretation may be hampered by false negative results or by artifacts due to the presence of polymorphisms generating abnormal restriction patterns [Nakagawa et al, 2003]. Semiquantitative PCR protocols, which are more practical than FISH and Southern blot for routine applications, include semiquantitative multiplex PCR of short fluorescent fragments (QMPSF) and multiplex ligation-dependent probe amplification (MLPA) assays successfully adapted to the analysis of different genes [Charbonnier et al, 2000;Casilli et al, 2002;Schouten et al, 2002;Sellner and Taylor, 2004]. QMPSF is based on the simultaneous amplification of short genomic sequences that correspond to the different exons by fluorescently labeled primers, and several multiplex PCRs may be necessary to cover one gene [Charbonnier et al, 2000[Charbonnier et al, , 2002Wang et al, 2002;Casilli et al, 2002;Audrézet et al, 2004;Tournier et al, 2004].…”

“…In principle, the results obtained by applying a single semiquantitative method could be correctly interpreted when two or more contiguous exons are involved, since undesired mismatches are unlikely to simultaneously affect multiple oligonucleotides. Conversely, it is necessary that single exon deletions be verified by two independent methods [Taylor et al, 2003;Wehner et al, 2005;Sellner and Taylor, 2004]. Breakpoint characterization allows unequivocal confirmation of rearrangements and provides a PCR-based diagnostic tool to search for the mutation in at-risk family members.…”

Performance of MLPA as a screening method for aneuploidy in uncultured amniocytes

“…In MLPA, amplification of probes by PCR depends on the presence of small specific target sequences in the sample. Nucleotide mismatches at the probe binding site prevent probe hybridization and ligation and therefore single base changes may result in deletions (7,18). Therefore, in these variations, the relative signal ratios of the other probes specific to the related chromosome should be analyzed in detail.…”

Genome-wide gene expression profiling and mutation analysis of Saudi patients with Canavan disease

“…21 Briefly, in each reaction 250 -300 ng of genomic DNA was denatured and hybridized with the SALSA® probe-mix. After probe hybridization, a ligation reaction was performed using ligase-65 enzyme and subsequent products were amplified by 35 cycles of PCR; 30 seconds at 95°C, 30 seconds at 60°C, 1 minute at 72°C, and a final extension step of 20 minutes at 72°C.…”