HRM Introduction یکشنبه شانزدهم مرداد ۱۳۹۰

HRM Instrumentation

For several years, various researchers and instrument makers have independently investigated the utility of high-resolution DNA dissociation analysis. For example, the team at Idaho Technology has done an admirable job of vigorously promoting their research through traditional journal publications. Conversely, Corbett Life Science does not pursue publication, but instead relies on the publications of customers to promote the technology. Regardless, both companies have independently advanced the field of high resolution dissociation analysis and successfully introduced what has now become known as high resolution melt (HRM) analysis.
Idaho Technology was first to market with an instrument made specifically to do dissociation analysis; the HR-1. The HR-1 was a showpiece for the technology with the singular aim of producing the most detailed melt curve possible. As such, it opened the eyes of many to the potential of HRM and remains the performance benchmark for the acquisition of an individual melt curve. However the HR-1 is not capable of thermal cycling and can only analyze a single sample from within a glass capillary per run making data analysis time consuming.
Multi-well instruments with greater practical utility were introduced to the market very soon after the HR-1. The first multi-well HRM instruments were the Rotor-Gene 6000 (Corbett Life Science) and the LightScanner (Idaho Technology) (PDF). These two instruments were introduced at about the same time but employed fundamentally different technical innovations to achieve HRM. The LightScanner uses a modified block-based design available in 96-well or 384-well versions. Despite advanced engineering, it still suffers from measurable sample-to-sample thermal and optical variation and is unable to match the performance benchmark set by the original HR-1 instrument. Like the HR-1, the LightScanner is not capable of thermal cycling.
The Rotor-Gene 6000 was the first of the multi-well instruments capable of both thermal cycling and HRM. This dual capability enables samples to be fully processed in the one instrument (i.e. pre-amplification and HRM done consecutively in the one run). A major advantage of this is that amplification plots can be used to help interpret HRM results since aberrant amplification plots (i.e. those that amplified differently to what was expected) also produce aberrant HRM data. In this way compromised samples can be easily identified and removed from downstream HRM analysis. The main advantage of the Rotor-Gene for HRM stems from its rotary design, in which samples spin under centrifugal force past a common optical detector. This is seemingly ideal for HRM as thermal or optical variation between samples is insignificant. The result is that the Rotor-Gene HRM performance closely matches the HR-1 benchmark with the compromise that samples are not arranged in a conventional array format (as they are in block-based instruments) but are instead arranged around the perimeter of a spinning rotor.
The more recently introduced LightCycler 480 (Roche Molecular Systems) is capable of HRM and thermal cycling. The LightCycler 480 is a block-based instrument design and it has better thermal uniformity than other block-based instruments, it nevertheless does exhibit measurable thermal and optical non-uniformity.
Other instrument providers are now rushing to introduce HRM capability and some are planning to release software upgrades to support HRM analysis. The danger here is that instruments not specifically engineered for HRM will deviate so much from the HR-1 performance benchmark that careful investigation will need be done before accepting those instruments as HRM capable.

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