Microchip Electrophoresis for Detection of Circle-to-Circle Amplification Products towards Sensitive and Rapid DNA Analysis Laili Mahmoudian, 1 Jonas Melin, 2 Mohamad Reza Mohamadi, 1 Keiko Yamada, 3 Michio Ohta, 3 Noritada Kaji, 1;4 Manabu Tokeshi, 1;4 Mats Nilsson, 2 and Yoshinobu Baba 1;4;5;6 1 Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603 2 Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Se-75185, Uppsala, Sweden 3 Department of Molecular Bacteriology, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 4 MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603 5 Plasma Nanotechnology Research Center, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603 6 National Institute of Advanced Industrial Science and Technology (AIST), Health Technology Research Center, Takamatsu 761-0395 (Received December 4, 2006; CL-061422; E-mail: h051105d@mbox.nagoya-u.ac.jp) A new method for fast and precise analysis of circle-to-cir- cle amplification (C2CA) products by microchip electrophoresis has been developed. Stable C2CA products were produced by applying a new enzymatic step to C2CA. Detection was carried out within 55s with RSD of migration time of 3.6% (n ¼ 6) en- abling reproducibility and high speed. A real sample of bacterial pathogen (V. Cholerae) at single nucleotide level was detected successfully based on this method. Microchip electrophoresis (-CE) has been developed into powerful technology for the detection of DNA samples and suggests high throughput ability since it enables high speed, low cost and eligibility for automation. 1–3 Circularizing oligonu- cleotide probes, so-called padlock probes have two-ends com- plementary to the target strand. 4 Upon a perfect hybridization, the two adjacent ends are connected by ligation (Figure 1a). Padlock probes are powerful detectors of single nucleotide var- iations and pathogen diagnosis since they ensure high specificity and excellent selectivity. 5 Circle-to-circle amplification (C2CA) is a precise and sensitive technique for signal amplification of circularized padlock probes (Figure 1b). 6 Techniques for detection of C2CA have far been limited to microarrays, real time monitoring using molecular beacons, denaturing acrylamide-gel electrophoresis and single-molecule detection. 6,7 Although all those techniques are quite accurate, they are not ideally suited for multiplex medical diagnostics. Although detection based on molecular beacons and single- molecule detection is quite rapid and no post-amplification process is required, both strategies require design and synthesis of additional probes for labeling, and the multiplexing capacity is limited by the number of spectrally separable fluorophores. Microarrays require extensive post-amplification handling and expensive labeling. Denaturing acrylamide gel electrophoresis, among those, is the most available technique but like other slab-gel electrophoresis techniques suffers from low analytical speed, high labor intensity, and the difficulty to automate. There- fore, it would be highly advantageous to develop a -CE-based detection system which enables a faster, cheaper, and technically simpler approach. This approach is based on widely available in- tercalating dyes which can be used in combination with simple -CE. Towards that end, producing a stable C2CA product for later analysis in a multiplexed format is indispensable. As a model sample, a species-specific padlock probe for detection of the 16S rRNA gene from V. Cholerae (a causative agent for acute human diarrhea) was used in this study. The pad- lock probe, replication oligonucleotide, and target sequences were as described by Jarvius et al. 7 (Supporting Information; SI 1). 10 The padlock probe was phosphorylated and circularized using a 39 bp synthetic target and T4 DNA ligase. The amplifi- cation proceeded through first replication and monomerization steps of the C2CA process. To make dsDNA C2CA products, an additional enzymatic step was applied to monomerized products using Phi29 DNA polymerase. Analysis of 100 nM products on an Agilent 2100 Bioanalyzer’s glass microchip yielded dsDNA products as well b: C2CA 5’ Ligation Replication 5’ 3' Duplication (Klenow fragment) Target complementary parts Padlock hybridization to the target Concatemer 3’ Template Padlock probe 3’ 3’ 5’ 5’ 3’ RO+ 3’ 5’ 5’ Transient open circle Monomerization (restriction digestion) a c Figure 1. DsDNA C2CA production mechanism (a) padlock probe-target hybridization (b) C2CA of circularized padlock probe. C2CA consists of three steps of ligation, replication, and monomerization. Monomerization means cutting the canca- temer products using restriction enzymes (c) the added final step to convert ssDNA products to dsDNA and the mechanism of in- crease in size of the produced dsDNA during the final step using Klenow fragment which displaces 5 0 end of attached RO+ and mediates synthesis of dsDNA C2CA products with blunt ends. RO+ refers to replication oligonucleotide with positive polarity. (Dahl et al.) 6 396 Chemistry Letters Vol.36, No.3 (2007) Copyright Ó 2007 The Chemical Society of Japan