Quantitation of In Situ Hybridization Analysis

Abstract

In situ hybridization is a powerful method for detecting specific DNA and RNA sequences in cells and tissue sections. Major areas of applications include cytogenetics, microbiology, and studies of gene expression and regulation. The methods are based on the fact that the purine and pyrimidine bases that make up DNA (adenine, A; guanine, G; thymine, T; and cytosine, C) or RNA (A, G, C, and uracil, U) can form specific base pairs (A-T, A-U, C-G) that link two complementary strands of DNA or RNA to each other. Thus, provided the sequence of interest (target) is available or known, it is possible to synthesize and label the complementary sequence and use it as a probe for in situ hybridization detection of the target. Since the reaction will contain only four (DNA-DNA, RNA-RNA) or, at most, five (DNA-RNA) bases as reactants, it is possible to define conditions mathematically under which only totally complementary sequences will hybridize. The strength of the hybridization is temperature dependent, and the melting point T m defines the temperature at which half the hybrids formed will dissociate or “melt.” The T m depends on the degree of complementarity (lack of mismatches), the number of bases (length of the probe), the frequency of GC base pairs (which bind more strongly than A-T or A-U), and the ionic strength. Addition of formamide is often used to lower the T m .