Protocol

Temperature Gradient Capillary Electrophoresis (TGCE) Assay

¹ Department of Agronomy, Iowa State University, Ames, IA 50011, USA
² Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011, USA
³ Center for Plant Genomics, Iowa State University, Ames, IA 50011, USA

⁴Corresponding author (schnable{at}iastate.edu)

INTRODUCTION

This protocol describes the use of temperature gradient capillary electrophoresis (TGCE) to test for polymorphisms between two DNA fragments. Polymorphisms may be detected within an individual or between individuals depending on the experimental design. In this assay, alleles of interest and a "reference" allele are PCR-amplified with high-fidelity Taq polymerase, and the resulting amplicons are then mixed in a 1:1 ratio. This mixture is denatured and reannealed to allow formation of homoduplexes (in the absence of polymorphisms between the allele of interest and the reference allele) and heteroduplexes (in the presence of polymorphisms). The Reveal System (SpectruMedix), which can analyze four 96-well plates at a time, is then used for electrophoresis and detection, and the data are scored and viewed with Revelation software.