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).

This image illustrates the process of DNA hybridization using oligonucleotide probes. On the left, there is a set of multicolored vertical bars representing unhybridized DNA strands, with each color representing different sequences. These are set to be combined with a surface that has numerous short blue bars sticking out, labeled as oligonucleotide probes. An arrow points to an image in the middle, the DNA and probes are combined on the surface, suggesting the preparation for hybridization. Another arrow from the middle points to the right, the image shows the result of hybridization: some of the DNA strands have bound to complementary oligonucleotide probes on the surface, indicated by the matching colors between the DNA strands and the probes. One of the hybridized DNA strands has a red spot representing a marker or label for detection.

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).