title: Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system.
abstract: BACKGROUND
  With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target.
  
  
  METHODS
  We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses.
  
  
  RESULTS
  RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls).
  
  
  CONCLUSIONS
  The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.
authors.affiliation: Eragen Biosciences, Inc., 918 Deming Way, Madison, WI 53717-1944, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Moser
authors.forename: David J
authors.initials: DJ
authors.lastname: Marshall
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Grenier
authors.forename: Collin D
authors.initials: CD
authors.lastname: Kieffer
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Killeen
authors.forename: Jerod L
authors.initials: JL
authors.lastname: Ptacin
authors.forename: Craig S
authors.initials: CS
authors.lastname: Richmond
authors.forename: Eric B
authors.initials: EB
authors.lastname: Roesch
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Scherrer
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Sherrill
authors.forename: Cris V
authors.initials: CV
authors.lastname: Van Hout
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Zanton
authors.forename: James R
authors.initials: JR
authors.lastname: Prudent
mesh_terms: D000199:Actins / Q000235:genetics; D001482:Base Composition; D004735:Energy Transfer; D005165:Factor V / Q000235:genetics; D005838:Genotype; D006801:Humans; D009154:Mutation; D009696:Nucleic Acids / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D011516:Prothrombin / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000199:Actins; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin