Nonspecific Chemistries

An important advantage of nonspecific chemistries is that the design and setup of the assays is straightforward and, except for the cost of the real-time PCR apparatus, material costs are low. This makes them particularly attractive for the analysis of single nucleotide polymorphisms (SNP), which have become the markers of choice for identifying the multiple genes associated with complex diseases such as cancer or diabetes. Biallelic polymorphisms can be detected by combining allele-specific amplification with the detection of SYBR Green I. Allele-specific amplification takes advantage of the relative inability of Taq polymerase to extend primers that are mismatched to their targets at the 3' end. The assay is carried out in two separate tubes, each of which contains a primer pair specific to one or the other allelic SNP variant.[2] Although there will be amplification of the mismatched allele, this occurs much less efficiently than that of the matching allele, delaying amplification and resulting in a much higher Ct being recorded. The specificity of the assay can be improved by using hairpin primers for the allele-specific PCR, as they are better than linear ones at discriminating between closely related

sequences.[3]

The use of dissociation curve analysis to identify different amplicons obviates the requirement for two

Fig. 1 Schematic representation of a real-time PCR assay using MB probes. At the annealing temperature, MB bind to their complementary target and fluorescence readings are taken. At the polymerization temperature MB dissociate from their target allowing Taq polymerase to read through and replicate the amplicon. A well-designed assay has an amplification efficiency of near 100% at the time fluorescence is first detected. Hence, any twofold difference in initial template concentration is reflected in a DCt of 2.

Fig. 1 Schematic representation of a real-time PCR assay using MB probes. At the annealing temperature, MB bind to their complementary target and fluorescence readings are taken. At the polymerization temperature MB dissociate from their target allowing Taq polymerase to read through and replicate the amplicon. A well-designed assay has an amplification efficiency of near 100% at the time fluorescence is first detected. Hence, any twofold difference in initial template concentration is reflected in a DCt of 2.

separate amplification reactions. Following the PCR assay, double-stranded (ds) DNA product is melted into single-stranded (ss) DNA by a stepwise increase in temperature, with fluorescence data being collected at each temperature step. The magnitude of the reduction in fluorescence intensity of the SYBR Green dye due to its release from dsDNA provides an indicator of the amount of dsDNA dissociated at each step in the dissociation curve (Fig. 3). Furthermore, as different amplicons will melt at different temperatures, SYBR Green I can be used to distinguish different alleles through their melting temperatures (Tm). For example, Huntington's disease is caused by an expanded number of CAG repeats in the Huntington gene and the dissociation curve of a normal subject shows a single melting peak, whereas that of an affected individual has two.[4] Several closed-tube systems have been developed that can be used in combination with melting analysis of PCR products to identify both heterozygous and homozygous sequence variants,[5] and it has even been possible to develop triplex assays that use

SYBR Green I and dissociation curves to identify different gene targets in the same tube.[6]

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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