In a reverse SSO assay, the genomic DNA is often incubated with sodium hydroxide before application to the membrane. Why?

The process of incubating genomic DNA with sodium hydroxide in a reverse SSO (Sequence-Specific Oligonucleotide) assay is primarily to denature the DNA. Denaturation involves the separation of the two strands of DNA, which is essential for the hybridization process that follows. By breaking the hydrogen bonds between the base pairs, sodium hydroxide creates single-stranded DNA that allows for better accessibility for the oligonucleotide probes on the membrane. This accessibility is crucial for the specific binding of the probes to their complementary targets, maximizing the assay's sensitivity and accuracy.

In a reverse SSO assay, having single-stranded DNA is vital as it facilitates the hybridization of the specific oligonucleotide probes used to detect particular sequences. If the DNA were not denatured, the double-stranded structure would impede the effective binding of oligonucleotide probes, leading to poor assay performance and potentially false results. All these factors underline the importance of DNA denaturation in ensuring effective detection in genomic analysis.