Temperature (°C)

Fig. 2 Rapid analysis of sickle cell anemia polymorphisms. (A) The HyBeacon probe employed to genotype samples with respect to sickle cell disease was fully complementary to the sickle cell (HbS) allele, exhibiting single and double nucleotide mismatches when hybridized to normal b globin gene (HbA) and hemoglobin C (HbC), respectively. (B) HyBeacon probe hybridized to the fully complementary HbS allele possesses a Tm of approximately 58°C. Hybridization to the mismatched HbA and HbC alleles drops the probe Tm to 52°C and 48°C, respectively. Combinations of probe melt peaks allow determination of HbA/A, HbS/S, HbA/S, and HbA/C genotypes.

LightCycler. HyBeacon probes are capable of detecting both DNA and RNA targets, being compatible with single-tube RT-PCR, and PCR assays may comprise both three- and two-stage cycling protocols. Probes and assays may be designed such that HyBeacons are not hybridized to target sequences during PCR, thereby avoiding obstruction of DNA polymerase (cf. TaqMan) and enabling the use of extremely rapid thermal cycling conditions. Forty-five cycles of amplification have been achieved in less than 10 min using two-stage LightCycler thermal protocols and short incubation times. Following amplification, melt analysis may be completed in as little as 2 min. The length and composition of target sequences will affect amplification efficiency and determine PCR cycling conditions and assay duration. Although target sequences of 80-150 bp are typically employed in HyBeacon assays, it is sometimes necessary to analyze larger amplicons to ensure specificity in the presence of closely related sequences such as pseudo-genes. The type and purity of samples to be analyzed will also affect the optimal PCR conditions and test duration.

Saliva samples may be employed directly in HyBeacon assays without the requirement for DNA purification.1-2-1 Direct saliva analysis not only reduces the overall duration of assays, but also makes the tests noninvasive, thereby avoiding the safety issues associated with diagnostics using blood samples. Furthermore, providing saliva samples is easier, more acceptable, and less stressful for patients. Ultrarapid analyses will permit HyBeacons to be employed in point-of-care diagnostic scenarios and in-the-field applications where results are required quickly.

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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