Pyrosequencing for SNP Genotyping

Abstract

Pyrosequencing is a new DNA sequencing technique based on sequencing-by-synthesis (1). This technique enables real-time detection using an enzyme-cascade system, consisting of four enzymes and specific substrates, to produce light whenever a nucleotide forms a base pair with the complementary nucleotide in a DNA template strand. As a result of nucleotide incorporation inorganic pyrophosphate (PPi) is released and is subsequently converted to ATP by ATP sulfurylase which is used by luciferase to generate proportional amount of light. Unreacted nucleotides are degraded by the enzyme apyrase, allowing iterative addition of nucleotides (see Fig. 1). DNA template generated by PCR is hybridized with a sequencing primer prior to Pyrosequencing. Using one pmol of DNA, 6×10¹¹ ATP molecules can be obtained per nucleotide incorporation which, in turn, generate more than 6×10⁹ photons at a wavelength of 560 nanometers. This amount of light is easily detected by a photodiode, photomultiplier tube, or a CCD-camera. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and simple automation. Furthermore, the technique avoids the use of labeled primers, labeled nucleotides, and gel-electrophoresis.