title: Single-Tube Mutation Scanning of The Epidermal Growth Factor Receptor Gene Using Multiplex LATE-PCR and Lights-On/Lights-Off Probes.
abstract: BACKGROUND
  Numerous mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene determine the response of many patients with non-small cell lung carcinoma (NSCLC) to anti-EGFR tyrosine kinase inhibitors (TKIs). This paper describes a single closed-tube assay for simultaneous mutational scanning ofEGFRexons 18-21.
  
  
  METHODS
  The assay first co-amplifies all four exons as separate single-stranded DNA products using Linear-After-The-Exponential (LATE)-PCR. The amplicons are then interrogated at endpoint along their length using sets of Lights-On/Lights-Off probes of a different color for each exon. The four resulting fluorescent signatures are unique for each underlying DNA sequence. Every mutation in a target potentially alters its unique fluorescent signature thereby revealing the presence of the mutation.
  
  
  RESULTS
  The assay readily detects mutations which cause sensitivity or resistance to TKIs and can distinguish these clinically important genetic changes from silent mutations which have no impact on protein function. The assay identifies as little as 5% mutant sequences in mixtures of normal DNA and mutant DNA prepared from cancer cell lines. Proof-of-principle experiments demonstrate mutation identification in formalin-fixed, paraffin-embedded NSCLC biopsies.
  
  
  CONCLUSIONS
  The LATE-PCREGFRassay described here represents a new type of highly informative, single-tube diagnostic test for mutational scanning of multiple gene coding regions and/or multiple gene targets for personalized cancer therapies.
authors.affiliation: Department of Biology, Brandeis University, 415 South St., Waltham, MA, USA.
authors.forename: Shana M
authors.initials: SM
authors.lastname: Tetrault
authors.affiliation: Department of Biology, Brandeis University, 415 South St., Waltham, MA, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Rice
authors.affiliation: Department of Biology, Brandeis University, 415 South St., Waltham, MA, USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Wangh
authors.affiliation: Department of Biology, Brandeis University, 415 South St., Waltham, MA, USA.
authors.forename: J Aquiles
authors.initials: JA
authors.lastname: Sanchez
keywords: EGFR gene mutation scanning; LATE-PCR; Lights-On/Light-Off probes; molecular diagnostics; personalized cancer medicine; single-tube assay