Analyzing Genetic Variation
When double-stranded DNA (dsDNA) is heated, it unwinds into single-stranded counterparts. This property is integral to any PCR reaction. However, dissociation of dsDNA is also used in high-resolution melting (HRM) analysis. In this case, an amplicon is first generated to the area of interest by PCR in the presence of a dye that only emits light when bound to dsDNA. As the temperature is increased, the fluorescence decreases, creating a melting curve in real time that can be compared to a standard sequence to identify subtle genetic variations.
“The high precision melting of PCR products depends on several parameters such as base composition and distribution, as well as overall amplicon length,” explained Andreas Missel, Ph.D., associate director of R&D at Qiagen. “The melting curve delivers a characteristic profile, allowing resolution of fine differences for analyzing genetic variations such as for SNP genotyping, mutation screening, and DNA-methylation patterns.”
There are a number of factors that are critical to the success of HRM. “Reliable HRM analysis requires a suitable HRM instrument, the proper chemistry, and specific data-analysis software.” Dr. Missel noted. “There is also a stringent requirement for temperature uniformity in instruments. Often there can be subtle temperature differences within the qPCR thermocycler that significantly affect results.
“Another problem is that preparations done with ‘home-brew’ DNA isolations contain contaminants such as salts, phenol, or ethanol that alter curves. Additionally, data interpretation can be a challenge. There is still some room for improvement in this area. Often the HRM analysis software that comes with a PCR instrument requires some arbitrary user intervention.”
To streamline the process, Qiagen offers tools such as the Type-it HRM PCR and EpiTect HRM PCR kits that do not require optimization, and a thermocycler with accompanying software. “In our thermocycler, the Rotor-Gene Q, each tube spins in a chamber of moving air, keeping all samples at precisely the same temperature during rapid thermal cycling. This provides the high consistency needed for applications such as HRM.”
The future of HRM analysis will likely be “quite interesting,” predicted Dr. Missel. “There were about 50 publications in the literature on HRM four years ago. In 2009, there were about 500. Therefore, users are exponentially increasing, and there will presumably be many more types of applications for HRM on the horizon.”
Utilizing qPCR for single-cell analysis remains a significant hurdle, according to Martina Reiter, Ph.D., CEO of BioEPS. “Although single-cell analysis is not new, we still don’t have a final solution as to how to handle it. The biggest problem is technical variance during the experimental steps, especially in sampling analysis and subsequently data analysis.”
“Often samples are generated from laser capture microdissection and flow cytometry sorting. Sample amounts are very small and variances are huge. When you don’t pay attention in the beginning to such variances, downstream processes are even more inaccurate.”
What about solutions? “We are at the beginning of working on this and are continuing to determine the right way to do it,” Dr. Reiter said. “Substantial improvements in the analysis steps are needed. You cannot use standard analysis methods; they need to be modified for single-cell qPCR. But it is still difficult to modify so that technical variances are reduced as much as possible. Despite this, qPCR technology is sensitive enough to detect specific genes out of one cell.”
The MIQE guidelines are a newly proposed set of suggestions to standardize and document qPCR. “MIQE does not cover specific single-cell analysis so far, and qPCR advances need to be made in documenting and determining RNA integrity, in how to handle the necessity of preamplification, in the ability to more accurately sample cells, and in downstream analysis methodologies.”
Recognizing the importance of tackling these issues now will especially impact the future of therapeutics. “Ultimately, these problems will be solved,” Dr. Reiter predicted. “For the present, we need to realize that while qPCR for single-cell analysis is possible, especially technically, the larger issues of experimental design and variation remain and should be addressed.”