Melting Curve Analysis on the Light Cycler

The new LightCycler PCR technology measures the match of a hybridization probe with the polymorphic region of DNA by melting curve analysis and allows simultaneous detection of both HPA alleles. The Light-Cycler (http://www.roche-applied-science.com/lightcy-cler-online/) is a microvolume fluorometer integrated with a thermal cycler that combines rapid thermal cycling for PCR with real-time fluorescence monitoring. First, PCR is performed with two hybridization probes which are labeled with different fluorophores at the adjoining ends. One probe is designed to hybridize to the polymorphic sequence of the PCR templates, and the other hybridizes one to five nucleotides apart on the adjacent region. When these two probes anneal to the PCR template, they are in close proximity, and fluorescence energy transfer (FRET) occurs between the two fluo-rophores. The emitted fluorescence measured by the LightCycler monitors the amplification process in real time. After the amplification is completed, melting curve analysis allows genotyping. For melting curve analysis, the temperature is lowered below annealing temperature and then slowly increased. At a certain temperature, the probes melt off the DNA template, causing decreased fluorescence. The melting point can then be determined using an algorithm. When probe and PCR product match completely, the melting point is relatively high. A single point mutation, however, makes hybridization weaker and results in a lower melting point. Thus alleles can be easily discriminated. HPA typing on the LightCycler has been accomplished for HPA-1 to -5 and HPA-15.[9-11]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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