Single Probe Multiplex Analysis

Real-time PCR probe technologies, such as TaqMan and Scorpions,[7-10] achieve target detection through

Fig. 1 Sequence analysis by Tm determination. HyBeacon fluorescence emission decreases as the temperature increases and probe/ target duplexes melt to become single stranded. The upper graph displays melting curves from a probe hybridized to a fully complementary target and a sequence containing a single nucleotide mismatch. Fully complementary probe/target duplexes are more stable than duplexes containing positions of mismatch and exhibit considerably higher Tms. The lower graph displays the melt data in peaks to clearly measure probe Tm and determine the nature of the target sequence. (View this art in color at www.dekker.com.)

Fig. 1 Sequence analysis by Tm determination. HyBeacon fluorescence emission decreases as the temperature increases and probe/ target duplexes melt to become single stranded. The upper graph displays melting curves from a probe hybridized to a fully complementary target and a sequence containing a single nucleotide mismatch. Fully complementary probe/target duplexes are more stable than duplexes containing positions of mismatch and exhibit considerably higher Tms. The lower graph displays the melt data in peaks to clearly measure probe Tm and determine the nature of the target sequence. (View this art in color at www.dekker.com.)

monitoring fluorescence emission during amplification. Increased levels of fluorescence are indicative of target amplification and may be employed to assess the presence or absence of particular sequences in test samples. However, numerous probes, labeled with spectrally distinct fluorophores, are typically required to simultaneously detect and differentiate multiple target sequences in a single reaction vessel. For example, different fluorescent probes are required for each allele of polymorphic genes to ensure correct identification of homozygous and heterozygous samples.

HyBeacon fluorescence typically only acquired postamplification during melting curve analysis. Determination of melt peak Tms allows multiple target sequences to be simultaneously detected and identified using a single probe and fluorophore (Fig. 2).[1'2'4] Target sequences differing by as little as a single nucleotide may be simultaneously detected and reliably differentiated through the generation of multiple melt peaks such that samples homozygous and heterozygous for specific SNPs may be reliably genotyped with a single probe. This single-fluorophore method of multiplex sequence analysis may also be employed for applications, such as pathogen detection, that require an endogenous amplification control. Furthermore, unrelated target sequences may be simultaneously detected using multiple probes labeled with the same fluorophore. The number of target sequences that may be simultaneously analyzed depends on the Tms of each probe/target duplex, where a difference of at least 5°C is required to separate melt peaks and ensure correct identification of sequences.1-1-1 The example presented below demonstrates simultaneous analysis of two adjacent polymorphisms in the human p globin gene, where combinations of three different melt peaks allow samples to be reliably genotyped.

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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