Introduction

Pyrosequencing is a high-throughput technique for single nucleotide polymorphism (SNP) genotyping and DNA sequencing that is inexpensive, accurate, and easy to optimize. The pyrosequencing technique uses a non-electrophoresis-based bioluminometric DNA sequencing method that employs a cascade of luciferase-based enzymatic system that monitors DNA synthesis in real time. In the pyrosequencing assay, a sequencing primer is hybridized to a single-stranded, PCR-amplified DNA template and is then incubated with enzymes and their substrates. During the DNA sequencing, one of the four deoxyribonucleotide triphosphates (dNTPs) is added to the reaction system in an order that is preprogrammed. If the nucleotide is complementary to the nucleotide in the template, DNA polymerase catalyzes the incorporation into the DNA strand. When an incorporation event occurs, it is accompanied by the release of an inorganic pyrophosphate (PPi) in an equimolar amount to the incorporated nucleotide. The inorganic pyrophosphate produced is then converted to ATP by sulfurylase and is immediately utilized in a subsequent luciferase-based light-producing reaction. The pyrosequencer detects the visible light generated by luciferase during DNA synthesis, which is proportional to the number of nucleotides incorporated. The DNA sequence is then determined by monitoring the visible light produced from the reaction as each dNTP is added. The unincorporated nucleotides are degraded by apyrase, which allows the iterative addition of nucleotides in the following steps.

. ATP sulfurylase

PPi + adenosine 5 phosphosulfate-> ATP

ATP + luciferin NNN!!; AMP + oxyluciferin + hv ATP + dNTP NN^ADP + dNDP + dNMP + Pi

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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