SNP genotyping
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Pyrosequencing 189 13 189 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 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 nucle- otide 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 × 10 11 ATP molecules can be obtained per nucleotide incor- poration which, in turn, generate more than 6 × 10 9 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, flexibil- ity, parallel processing, and simple automation. Furthermore, the technique avoids the use of labeled primers, labeled nucleotides,
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SNP genotyping
Transcript of SNP genotyping
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Pyrosequencing 189
13
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,
