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Molecular Pathology

DNA sequencing

Pyrosequencing


Author: Rodney E. Shackelford, D.O., Ph.D., Tulane University (see Reviewers page)
Revised: 13 February 2011, last major update February 2011
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.

General
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● Pyrosequencing is a real-time DNA sequencing method based on the detection of pyrophosphate (PPi) released during the incorporation of a complementary nucleotide in a DNA polymerization reaction

● The reaction occurs as follows:

                        DNA polymerase
(DNA)n + dNTP ------------------------------> (DNA)n+1 +PPi

                                                   Sulfurylase
PPi + adenosine 5’ phosphosulfate -------------------> ATP + SO4-2

ATP + luciferin + O2 ---------> AMP + PPi + CO2 + one photon

● The sequencing reaction begins with a sequencing primer binding to a complementary single-stranded DNA molecule to be sequenced
● DNA polymerase is added, and each dNTP is added and removed from the reaction
● When a base is added to the DNA being synthesized, PPi is released and subsequently converted to ATP by ATP sulfurylase in the presence of adenosine 5´ phosphosulfate
● The ATP is used in the conversion of luciferin to oxyluciferin, generating visible light proportional to the ATP concentration
● Therefore, the amount of light produced in each reaction is proportional to the number of deoxynucleotides incorporated and the DNA sequence is read by comparing the light emitted to the dNTP added to each reaction
● The light is usually measured by a photomultiplier tube, avalanche photodiode or charge-coupled device camera
● The key to this sequencing technique is removal of previously added dNTPs from the reaction, so that the effect of new dNTPs on light emission can be measured

Solid and liquid phase sequencing
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● Removal of previously added dNTPs is accomplished by both solid and liquid phase pyrosequencing
● In solid phase pyrosequencing, a three enzyme mix (DNA polymerase, ATP sulfurylase, firefly luciferace) is used, and the template is bound to a solid phase, such as a magnetic bead
● After completing each dNTP reaction, the template is washed to remove non-incorporated deoxynucleotides and ATP resulting from the sulfurylase reaction.
● In liquid phase sequencing, a fourth enzyme, such as apyrase, is added to degrade the unreacted nucleotides and ATP
● One problem with both solid and liquid phase pyrosequencing is the interference of the dATP in luminescence detection, because added dATP would react with luciferin to produce a photon. This problem was solved via replacing dATP with dATPαS - this allows efficient dATPαS incorporation by DNA polymerase while reducing photon production by luciferin, as dATPαS in not a substrate for this enzyme
● The addition of single stranded DNA-binding proteins to pyrosequencing reactions had been found to reduce nonspecific primer binding, thus reducing mispriming and increasing signal intensity, allowing for reading higher accuracy. Currently pyrosequencing allows 300-500 nucleotides stretches to be read, less than that of dideoxynucleotide chain-terminating

Comparison with other methods
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Advantages: does not require labeled primers or dNTPs or gel electrophoresis; is done in real-time and is cost-effective when compared to dideoxynucleotide chain-terminating sequencing methods
Disadvantages: eventual loss of signal in the solid phase protocol due to loss of template, and the eventual loss of apyrase activity in liquid phase sequencing due to the accumulation of intermediate products which inhibit this enzyme; the technique is less accurate with homopolymeric runs of over 5-6 nucleotides in a row
● Pyrosequencing is usually used to analyze secondary DNA structures, to detect mtuations, and to sequence short to medium length DNA segments
● Parallel pyrosequencing platforms have been developed, such as the 454 Life Science, which can sequence entire bacterial genomes in 10 hours, demonstrating that pyrosequencing has significant research and biotechnology applications

End of Molecular Pathology > DNA sequencing > Pyrosequencing


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