Matrix-assisted laser desorption/ionization detection of polymerase chain reaction products by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase

The 5′‐3′ exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). This method o...

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Bibliographic Details
Published in:Rapid communications in mass spectrometry 2003-01, Vol.17 (6), p.532-537
Main Authors: Isola, N. R., Liu, Z., Allman, S. L., Taranenko, N. I., Kong, Y., Chen, C. H.
Format: Article
Language:English
Subjects:
DNA - biosynthesis
DNA - genetics
DNA - metabolism
Exonucleases - metabolism
Humans
Multienzyme Complexes - metabolism
Polymerase Chain Reaction
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Taq Polymerase - metabolism
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Summary:The 5′‐3′ exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). This method obviates the need to perform extensive DNA purification of reaction products that is often necessary for detecting larger DNA molecules by mass spectrometry. Oligonucleotides complementary to the internal region of the amplicon are degraded by the 5′‐3′ exonuclease activity and the degradation products are analyzed by MALDI mass spectrometry. We refer to this assay as the Exo‐taq assay or probe degradation assay. This method should be amenable to automation. Copyright © 2003 John Wiley & Sons, Ltd.
ISSN:0951-4198
1097-0231
DOI:10.1002/rcm.939