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. 2012 Nov 19;51(47):11800-4.
doi: 10.1002/anie.201205112. Epub 2012 Oct 16.

Signal amplification by glyco-qPCR for ultrasensitive detection of carbohydrates: applications in glycobiology

Seok Joon Kwon  1 , Kyung Bok Lee, Kemal Solakyildirim, Sayaka Masuko, Mellisa Ly, Fuming Zhang, Lingyun Li, Jonathan S Dordick, Robert J Linhardt
Affiliations

Signal amplification by glyco-qPCR for ultrasensitive detection of carbohydrates: applications in glycobiology

Seok Joon Kwon et al. Angew Chem Int Ed Engl. .

Abstract

Tiny amounts of carbohydrates (ca. 1 zmol) can be detected quantitatively by a real-time method based on the conjugation of carbohydrates with DNA markers (see picture). The proposed method (glyco-qPCR) provides uniform, ultrasensitive detection of carbohydrates, which can be applied to glycobiology, as well as carbohydrate-based drug discovery.

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Figures

Figure 1
Figure 1
Ultrasensitive detection of carbohydrates using glyco-qPCR. a) Coupling of biotin hydrazide to the reducing end of a CS disaccharide in the presence of NaCNBH3, and subsequent attachment of DNA to the carboxy group of the nonreducing end of the biotinylated CS disaccharide using EDC/NHS. Colocalization and signal amplification is used to increase detection sensitivity of the target carbohydrate. b) Glyco-qPCR amplification plots of recovered BCS–DNA conjugates. Different amounts of BCS and trehalose as negative control were used to determe detection limitation. All samples and controls were amplified in triplicate.
Figure 2
Figure 2
Glyco-qPCR detection of CS disaccharides isolated from CHO cells. a) CE separation of CS disaccharides from different number of CHO cells. b–c) The qPCR analysis of the CS disaccharides–DNA conjugate fractionally collected by CE.
Figure 3
Figure 3
Carbohydrate–protein interaction assay using Glyco-qPCR. a) Glyco-qPCR protocols for heparin–AT interaction assay with heparin–DNA marker-I and chondroitin–DNA marker-II (negative control). b) Data analysis of Glyco-qPCR, exemplified for the detection of heparin–AT interaction. All samples and controls were amplified in triplicate.
Scheme 1
Scheme 1
Schematic representation of Glyco-qPCR. Target carbohydrates (GAGs) from biological samples can be conjugated with different DNA markers, followed by removing unreacted DNA and detecting the corresponding GAG–DNA conjugates with amplified signals (qPCR).
See this image and copyright information in PMC

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