Researchers in the Department of Pathology at the Hopkins School of Medicine suggest that High Resolution Melting (HRM) assays are simple and scalable methods for measuring HIV diversity. The HRM scores could be useful biomarkers to determine when HIV patients were infected.
HRM assays carry significant implications in the field of molecular biology, as they allow scientists to detect mutations or epigenetic changes in double-stranded DNA.
HRM analyses are performed on double stranded DNA. First, polymerase chain reaction, more commonly referred to as PCR, is conducted to amplify the DNA region in which the mutation resides. The amplified region is known as the amplicon. The HRM analysis requires very simple procedures; after the mutated region is amplified, the amplicon is heated until the double strands are pulled apart — the temperature at which this occurs is called the melting point.
Researchers use a unique fluorescent dye, known as intercalating dye, which only fluoresces when bound to double-stranded DNA. When the melting point of the amplicon is reached, the dye begins to dim, as more and more of the billions of PCR-produced DNA becomes single stranded. A camera attached to the HRM machine records and plots the data of the fluorescence into a graph known as a melt curve.
When a mutation occurs in a DNA region, the two strands of DNA are held together differently due to varied combinations of base-pairings. Although certain mutations can change the melting point by a very small fraction, the high resolution melting assays are able to detect these changes.
The degree of viral diversity in an HIV-infected individual can change due to immune and other selective pressures. The differences in HIV diversity can reflect the differences of viral dynamics, immune responses and HIV progression in certain individuals. The relatively novel method of determining melting points of certain viral DNA regions was used in 203 adults in two regions in the gag gene, one region in the pol gene and three in the env gene. The gag gene expresses polyproteins, pol expresses the reverse transcriptase and env expresses the envelope protein.
The HRM results have suggested that individuals who were infected with HIV for longer periods have manifested greater viral diversity than those who have recently been infected. HRM has proven to be a powerful as well as cost-effective tool, allowing for large-scale genome projects to be conducted through HRM analyses. Due to its simplicity, HRM assays can be performed in labs by non-geneticists.
