Technical Challenges

A technical challenge for pyrosequencing is the limited annealing specificity for the sequencing primers to the templates as the pyrosequencing assay is performed at 28 °C because of the low thermostability of firefly luciferase. Measures have been applied to minimize the background noise due to the nonspecific binding of the sequencing primers and the formation of 3'-end loops. Glycine betaine has been used to stabilize the luciferase so that the pyrosequencing assay can be performed at 37°C.[21] Single-stranded DNA-binding protein has also been tested to enhance specific hybridization between sequencing primers and templates, therefore reducing background noise and increasing the accuracy and intensity of the assays.[22] Increasing the annealing specificity would allow the pyrosequencing assay to be directly performed on double-stranded DNA and longer templates, therefore reducing the cost and time of assay.

Another disadvantage of pyrosequencing is the cost and time associated with purifying single-stranded DNA templates. A methodology has been developed to reduce the cost of generating biotin-labeled pyrosequenc-ing templates.1-5-1 This methodology uses a universal biotinylated primer and two sequence-specific primers. One of the sequence-specific primers contains a 5' tail that is complementary to the universal primer. During PCR amplification, a sequence-specific primer pair initiates PCR amplification of the genomic DNA. The resulting amplified fragments serve as templates for the universal biotinylated primer and a sequence-specific primer in subsequent amplifications, thus producing labeled ampli-cons. The cost savings result from ordering large amounts of the biotin-labeled universal primer rather than the biotin-labeled sequence-specific primers.

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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