Screening for imprinted genes using high-resolution melting analysis of PCR amplicons.
High-resolution melting (HRM) analysis is a technique that enables researchers to detect polymorphisms in DNA molecules based on different melting profiles and is becoming widely used as a method for detecting SNPs in genomic DNA. In this chapter, we describe how HRM analysis can be used to detect allelic imbalances typical of imprinted genes, where alleles are differentially expressed based on their parent of origin. This involves first producing hybrid seed using parental plants that have sufficient genetic differences to distinguish the parental origin of each allele of the candidate genes. RNA is then isolated from the hybrid seed and converted to cDNA. PCR amplicons are produced using primers designed to span a polymorphic sequence within the transcript of the candidate gene. By using a real-time PCR machine with HRM analysis capability, the PCR amplicons can be analyzed without further manipulations directly after amplification to detect instances of strong allelic imbalance and parent-of-origin-dependent expression.
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Robert
R
Day
Richard
R
Macknight
D000483:Alleles; D019295:Computational Biology / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018392:Genomic Imprinting; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D013223:Statistics as Topic; D044366:Transition Temperature
D017931:DNA Primers