title: Verification of multiplex ligation-dependent probe amplification probes in the absence of positive samples.
abstract: Deletions and duplications of single or multiple exons in specific genes are associated with human diseases. Multiplex ligation-dependant probe amplification (MLPA), a technique recently introduced to clinical laboratories, can detect deletions or duplications at the exon level. MLPA kits have a high multiplexing capability containing mixtures of exon-specific probes that target the gene of interest and control probes that hybridize to other genomic areas before PCR amplification. To verify each probe set, known positive samples with a single-exon deletion or duplication and normal samples are ideally used. Often, positive samples do not exist for each exon and normal samples are not suited to verify the identity of each probe set. We designed a straightforward approach using mixes of exon-specific PCR products as template to unequivocally verify each probe set in MLPA kits. This method can be used to verify the identity of MLPA probes for exons when positive samples are unavailable. Exon-specific probes from 15 MLPA kits were shown to hybridize to the targeted exons of interest. In one kit, this method identified probes that also bind a pseudogene, making them unreliable for clinical analysis. Incorporating this methodology in the analytical validation process will help ensure that MLPA results are interpreted correctly.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA.
authors.forename: Whitney
authors.initials: W
authors.lastname: Wooderchak-Donahue
authors.forename: Cecily
authors.initials: C
authors.lastname: Vaughn
authors.forename: Lan-Szu
authors.initials: LS
authors.lastname: Chou
authors.forename: Tracey
authors.initials: T
authors.lastname: Lewis
authors.forename: Kelli
authors.initials: K
authors.lastname: Sumner
authors.forename: Melinda
authors.initials: M
authors.lastname: Procter
authors.forename: Friederike
authors.initials: F
authors.lastname: Gedge
authors.forename: Pinar
authors.initials: P
authors.lastname: Bayrak-Toydemir
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
authors.forename: Genevieve
authors.initials: G
authors.lastname: Pont-Kingdon
mesh_terms: D015342:DNA Probes / Q000737:chemistry*; D005091:Exons; D015894:Genome, Human; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011933:Reagent Kits, Diagnostic / Q000592:standards; D017384:Sequence Deletion
chemical_list: D015342:DNA Probes; D011933:Reagent Kits, Diagnostic