PPARGC1A and PPARG genotyping: Beginner's guide to genotyping with unlabeled probes

Abstract

Amplicon melting and genotyping with unlabeled probes has become wide spread technique for SNP genotyping. In both methods PCR is performed with new generation of double-strand DNA binding dyes. Nucleic acid melting generates distinct melting curves that can be used to identify the presence of sequence variation within the amplicon. Unlabeled probes are inexpensive and provide the sequence specificity. This chapter provides comprehensive instructions about design of primers and probes and evaluation of their quality. Chapter also contains useful advices on how to perform DNA purification, optimize PCR conditions, how to prepare working solutions, and how to interpret the data of melting analysis. © 2013 Springer Science+Business Media New York.