Melting Temperature Analysis

A melting temperature (Tm) analysis measures the dissociation rates of hybridized nucleic acids, and there are two main methods by which this technique can be used to differentiate PCR products. Either the dissociation of a specific probe molecule from a target strand can be analyzed[8] or the temperature at which an entire amplicon molecule denatures can be determined.[6] When using specific probes (hybridization probes or minor groove binding probes), Tm discrimination is achieved based on the hybridization affinities of the probes for the target amplicon. Following thermocycling, the probes are allowed to anneal to the target amplicons. Upon hybridization of the probes to their specific targets, fluorescence is produced. The reaction temperature is then gradually increased, and fluorescence emissions are continually monitored. When a probe reaches its Tm, it dissociates from the amplicon and fluorescence ceases. Probes used for multiplexing can be designed with different lengths and different GC contents such that each has a distinguishable melting temperature. Besides being used to differentiate completely different targets, Tm analysis using specific probes can also be used to interrogate single nucleotide polymorphisms (SNPs). Targets with a single base pair mismatch with a probe sequence can readily be distinguished from targets with complete homology to the probe. The combination of colorimetric differentiation with Tm analysis creates the potential for multiplex detection and analysis of a large number of SNPs in a single reaction.

Melting temperature (Tm) analysis that does not involve the use of specific probes is based on the Tm of an entire double-stranded amplicon. In this case, fluo rescence is a product of molecules such as SYBR green that bind to double-stranded DNA and fluoresce when they are bound to the DNA double helix. Therefore at relatively low temperatures that permit DNA duplexes to remain stable, fluorescence will be observed, but when temperatures are increased and the double-stranded DNA molecules denature, the SYBR green will no longer bind to the nucleic acids, and fluorescence is no longer detectable. This strategy for Tm analysis will produce a characteristic melting curve that is based on the length, sequence, and GC content of each amplified product. However, it should be noted that using SYBR green lacks specificity because sequence-specific probes are not utilized. Consequently, genetic variability of target sequences from different patients makes identification of an amplified product, based on Tm of the amplicon, somewhat inconsistent.

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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