title: An efficient technique for primer development and application that integrates fluorescent labeling and multiplex PCR.
abstract: OBJECTIVE
  Development of genetic markers can be costly and time-consuming, especially when multiple primer pairs are fluorescently labeled. This step was streamlined by combining two techniques in the same PCR reaction: (1) custom-labeling of primers by the investigator and (2) multiplexing multiple primers together in the same reaction. •
  
  
  RESULTS
  This technique was successfully used to develop microsatellite markers in several plant species. Microsatellites amplified with this multiplexing process were identical to those generated from PCR using individual primer pairs and with traditional methods using a priori labeled fluorescent primers. Tests of PCR cycling programs revealed that conditions recommended for the commercial kit generated stronger fragment peaks than the previously recommended cycling protocol. •
  
  
  CONCLUSIONS
  This technique is an efficient and economical way to fluorescently label multiple microsatellite primers in the same reaction. It is also applicable to other markers used in PCR amplification of genetic material.
authors.affiliation: Department of Biological Sciences, University of Cincinnati, 614 Rieveschl Hall, Cincinnati, Ohio 45221-0006 USA.
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Culley
authors.affiliation: Department of Entomology, Purdue University, 901 West State Street, West Lafayette, Indiana 47907-2089 USA.
authors.forename: Trevor I
authors.initials: TI
authors.lastname: Stamper
authors.affiliation: Department of Biological Sciences, University of Cincinnati, 614 Rieveschl Hall, Cincinnati, Ohio 45221-0006 USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Stokes
authors.affiliation: Department of Biology, University of Kentucky, 101 T. H. Morgan Building, Lexington, Kentucky 40506 USA.
authors.forename: Jessica R
authors.initials: JR
authors.lastname: Brzyski
authors.affiliation: University of Arkansas, 534 West Research Center Boulevard, ENTR 120, Fayetteville, Arkansas 72701 USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Hardiman
authors.affiliation: Biology Program, Centre College, 600 West Walnut Street, Danville, Kentucky 40422 USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Klooster
authors.affiliation: Department of Biological Sciences, University of Cincinnati, 614 Rieveschl Hall, Cincinnati, Ohio 45221-0006 USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Merritt
keywords: fluorescent labeling; microsatellites; multiplexing; primer testing; thermocycler conditions