Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene

Nucleic Acids Res. 2004 Oct 11;32(18):e139. doi: 10.1093/nar/gnh137.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromatography, Liquid*
  • DNA Mutational Analysis / methods*
  • Exons
  • Gene Dosage
  • Gene Duplication
  • Genes, Retinoblastoma*
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Retinoblastoma / genetics
  • Sequence Deletion