HYBRIDIZATION ON DNA MICROARRAYS
Hybridization on a microarray is similar to the hybridization of DNA during other hybridization experiments, such as Southern blots, Northern blots, or dot blots. All these techniques rely on the complementary nature of double-stranded DNA. When two complementary strands of DNA are near each other, the bases match up with their complement, that is, thymine with adenine, and guanine with cytosine. On a DNA microarray, hybridization is affected by the same parameters as in these other techniques.
How the DNA is attached to the slide can affect how well the probe DNA and target DNA hybridize, especially for oligonucleotide microarrays (Fig. 8.22). The short length of oligonucleotides requires that the entire piece be accessible to hybridize. The length of the spacer between the oligonucleotides and the glass slide optimizes hybridization. An oligonucleotide attached with a short spacer has many of its initial nucleotides too close to the glass and inaccessible to incoming RNA or DNA. Oligonucleotides with longer spacers may fold back and tangle up; therefore, again the sequence is inaccessible for hybridization. Oligonucleotides attached with medium-sized spacers are far enough from the glass, but not so far as to get tangled. Thus medium-sized spacers give the best accessibility for hybridization.
Hybridization of two lengths of DNA (or RNA with DNA) requires certain sequence features. One important property is the relative number of A:T base pairs versus G:C base pairs. Because G:C base pairs have three hydrogen bonds holding them together, it takes more energy to dissolve the bonds. A:T base pairs have only two hydrogen bonds and require less energy. Thus more GC base pairs give stronger hybridization. If the sequence has many A:T base pairs, the duplex may form slowly and be less stable. Another important consideration is secondary structure. If the probe sequence can form a hairpin structure, it will hybridize poorly with the target. If the probe has several mismatches relative to the target, the duplex may not form efficiently. All these issues must be addressed when making an oligonucleotide microarray. Computer programs are available that identify suitable regions of genes with sequences that will produce effective probes.
cDNA arrays are less prone to the problems seen in oligonucleotide arrays. cDNAs are double- stranded, so secondary structures such as hairpins are less likely to be a problem. During a hybridization reaction, cDNA arrays must be denatured either with heat or chemicals, making the probes single-stranded. Then the single-stranded RNA samples are allowed to hybridize on the slide under conditions that promote duplex RNA:cDNA without any mismatches.