Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene.
Screening for large gene rearrangements is established as an important part of molecular medicine but is also challenging. A variety of robust methods can detect whole-gene deletions, but will fail to detect more subtle rearrangements that may involve a single exon. In this paper, we describe a new, versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and quantitated by fluorescent detection using a post-column intercalation dye. The relative peak intensities for each target directly reflect exon copy number. This novel technique was used to screen a panel of 121 unrelated retinoblastoma patients who were tested previously using a reference strategy. MP/LC correctly scored all deletions and demonstrated a previously undetected RB1 duplication, the first to be described. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to denaturing HPLC (DHPLC) users since it broadens the spectrum of available applications on a DHPLC system.
Service de Génétique Oncologique, Pathologie Moléculaire des Cancers, Institut Curie, Paris, France.
C
C
Dehainault
A
A
Laugé
V
V
Caux-Moncoutier
S
S
Pagès-Berhouet
F
F
Doz
L
L
Desjardins
J
J
Couturier
M
M
Gauthier-Villars
D
D
Stoppa-Lyonnet
C
C
Houdayer
D001483:Base Sequence; D002853:Chromatography, Liquid; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D018628:Gene Dosage; D020440:Gene Duplication; D016161:Genes, Retinoblastoma; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012175:Retinoblastoma / Q000235:genetics; D017384:Sequence Deletion