snp genotyping

Pyrosequencing 189

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189

From: Methods in Molecular Biology, vol. 212:Single Nucleotide Polymorphisms: Methods and Protocols

Edited by: P-Y. Kwok Humana Press Inc., Totowa, NJ

Pyrosequencing for SNP Genotyping

Mostafa Ronaghi

1. Introduction

Pyrosequencing is a new DNA sequencing technique based onsequencing-by-synthesis (1). This technique enables real-timedetection using an enzyme-cascade system, consisting of fourenzymes and specific substrates, to produce light whenever a nucle-otide forms a base pair with the complementary nucleotide in a DNAtemplate strand. As a result of nucleotide incorporation inorganicpyrophosphate (PPi) is released and is subsequently converted toATP by ATP sulfurylase which is used by luciferase to generateproportional amount of light. Unreacted nucleotides are degradedby the enzyme apyrase, allowing iterative addition of nucleotides(see Fig. 1). DNA template generated by PCR is hybridized with asequencing primer prior to Pyrosequencing. Using one pmol ofDNA, 6 1011 ATP molecules can be obtained per nucleotide incor-poration which, in turn, generate more than 6 109 photons at awavelength of 560 nanometers. This amount of light is easilydetected by a photodiode, photomultiplier tube, or a CCD-camera.Pyrosequencing has the potential advantages of accuracy, flexibil-ity, parallel processing, and simple automation. Furthermore, thetechnique avoids the use of labeled primers, labeled nucleotides,