Nucleic acid hybridization

Nucleic acid hybridization is a technique to analyze the DNA or RNA of a biological sample. It can be used for measuring expression levels or genotyping. Nucleic-acid hybridization is based on the specific binding (annealing) of two complementary single-stranded nucleic acid (DNA or RNA) molecules. In DNA, the base adenine always forms a bond to thymine, while guanine always forms a bond to cytosine. In RNA, thymine is replaced by uranine to form a base pair with adenine.

To perform nucleic acid hybridization analysis, DNA or RNA is first purified from the sample. Stable, double-stranded nucleic acid sequences are denatured and fragmented to form short single strands, which can bind to single-stranded oligonucleotides with a known sequence called probes. Unbound DNA or RNA is washed off and binding of the target DNA or RNA to the probe is detected by fluorescent, radioactive or chemiluminescent reporter labels. If necessary, sample DNA can be amplified by PCR prior to analysis (amplified DNA hybridization assay).

Figure 1: Nucleic acid hybridization: Single-stranded target DNA or RNA is added to the oligonucleotide probes (step 1). Target DNA or RNA binds to complementary probes (step 2). Unbound target DNA or RNA is washed off and complementary binding is detected (step 3).