Pyrosequencing

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Pyrosequencing for SNP Genotyping
Mostafa Ronaghi

1. Introduction
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,
From: Methods in Molecular Biology, vol. 212:
Single Nucleotide Polymorphisms: Methods and Protocols
Edited by: P-Y. Kwok Humana Press Inc., Totowa, NJ

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