39] Assaying differential ribosomal RNA gene expression with allele-specific probes

Abstract

Publisher Summary This chapter discusses the rapid identification and characterization of multigene family sequence variants, the design of allele-specific oligonucleotide probes, and procedures for the use of the oligonucleotide probes to analyze differential ribosomal RNA (rRNA) transcript levels. Variation in multigene families, such as rDNA may be identified by several methods. The most common method involves cloning and sequencing of specific variants present in the samples of interest. Even with the advent of rapid cloning and sequencing methods, this can be a time-consuming process when several polymorphisms or variable samples are of interest. Potential sequence polymorphisms may be most rapidly identified by screening restriction enzyme digests of rDNA by Southern blot/hybridization mapping experiments. These restriction enzyme polymorphisms are described as potential sequence polymorphisms because in many cases in higher plants (and vertebrates), restriction enzyme polymorphisms that appear to be site losses are caused by methylation of cytosine residues (or adenine in some plants), thus, rendering the sequence refractory to cleavage by methylation-sensitive restriction enzymes.