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Nucleic Acids Research Advance Access originally published online on November 28, 2006
Nucleic Acids Research 2007 35(1):e3; doi:10.1093/nar/gkl939
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Nucleic Acids Research, 2007, Vol. 35, No. 1 e3
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer

Kenji Kinoshita1,3,*, Kentaro Fujimoto1, Toru Yakabe1, Shin Saito1, Yuzo Hamaguchi1, Takayuki Kikuchi2, Ken Nonaka2, Shigenori Murata2, Daisuke Masuda1, Wataru Takada1, Sohei Funaoka1, Susumu Arai1, Hisao Nakanishi1, Kanehisa Yokoyama1, Kazuhiko Fujiwara1 and Kenichi Matsubara2

1 Sumitomo Bakelite Co., Ltd. 1-1-5 Muroya, Nishi-ku, Kobe 651-2241, Japan 2 DNA Chip Research Inc. 1-1-43 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan 3 School of Pharmaceutical Sciences, Mukogawa Women's University 11-68 Kyuban-cho, Koshien, Nishinomiya 663-8179, Japan

*To whom correspondence should be addressed. Tel: +81 798 45 9982; Fax: +81 798 41 2792; Email: kenji_k{at}mukogawa-u.ac.jp

Received August 8, 2006. Revised October 1, 2006. Accepted October 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND FUTURE...
 SUPPLEMENTARY DATA
 REFERENCES
 
DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO® PrimeSurface® with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3' end of the immobilized DNA primers on the S-Bio® by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND FUTURE...
 SUPPLEMENTARY DATA
 REFERENCES
 
DNA microarrays have emerged as a powerful and promising revolutionary tool for large-scale parallel genetic analysis (1–3). Microarray fabrication can be accomplished using in situ light-directed combinatorial synthesis on the surface of arrays (4–6) or deposition methods (7), which are immobilized pre-synthesized oligonucleotides (8,9) on a solid support. Currently, oligonucleotide-based microarrays offer a number of advantages over cDNA microarrays and have global applications in gene expression profiling studies, genotyping such as of single nucleotide polymorphisms (SNPs) and resequencing (10,11), which have generally been performed by the hybridization of fluorescence-labeled oligonucleotides on a microarray.

Technologies such as photolithographic oligonucleotide DNA-directed synthesis allow the manufacture of high-density oligonucleotide microarrays, but are costly and time consuming thus limiting universal genome-wide investigation. Therefore, the immobilization of synthetic oligonucleotide probes is more common for many diagnostic tests by gene expression profiling. Recently, chemically pre-activated microscope glass slides have become commercially available, increasing the number of options for the covalent attachment of modified oligonucleotides to pre-activated glass slide supports (12–21).

As alternatives to these glass-based slides, plastic polymer slides, used in a few methods for the immobilization of oligonucleotides on microarrays, can be utilized to produce DNA microarrays. The advantages of DNA microarray fabrication on plastic-based substrates are that microarrays can be manufactured inexpensively in large numbers and easily formed into any size and shape such as miniaturized microarray devices or modules. As the development of DNA microarrays for plastics is still in its early stages, essential technologies for microarrays have been developed using polymer-based substrates such as poly(methyl methacrylate) (PMMA), and only recently have immobilization and hybridization been tested in a microarray-type format (22).

In this paper, we focus on the hybridization properties of a suitable surface chemistry for a cyclic olefin copolymer (COC) surface with random copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), and p-nitrophenyloxycarbonyl polyehyleneglycol methacrylate (MEONP) (23), and discuss new approaches for the application of an on-chip DNA detection method through Multiple Primer EXtension (MPEX) by DNA polymerase (Figure 1). DNA templates hybridize to solid surface-bound primers, which are then elongated with DNA polymerase to produce a copy of the hybridized template in the 5'->3' direction and deoxynucleotidyl triphosphates (dNTP) as extender units are incorporated into their nucleic acid chain elongation products. Also, a DNA amplification procedure similar to the PCR method on the solid surface occurs via the following mechanism. At each cycle, hybridization between DNA primers and the DNA templates present in the solution participates in the primer elongation process before being released back into the solution phase after denaturation at 95°C. The DNA templates repeatedly hybridize to attach neighboring primers to the solid surface and form additional fluorescent copies. In order to repeat this cycle, the primers must satisfy two main requirements. First, the surface density of the immobilized oligonucleotides should be high enough for template capture by hybridization after the cycle. Second, the covalent linkage between the oligonucleotide primer and the solid surface must be thermally stable under repeated heating/cooling cycles.


Figure 1
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  		Figure 1 Model for Multiple Primer EXtension (MPEX) reactions on the S-BIO® PrimeSurface®. The aminated DNA primers are 5' end covalently attached to the MPC surface of S-BIO® through the active ester moiety (MEONP). The MPEX reaction proceeded in the presence of template DNA, nucleotides (dNTP) and Taq DNA polymerase in a buffer on the S-BIO® PrimeSurface®.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND FUTURE...
 SUPPLEMENTARY DATA
 REFERENCES
 
Materials
All chemicals and solvents were purchased from Wako (Tokyo, Japan) or Sigma/Aldrich (Tokyo, Japan), unless stated otherwise, and used without additional purification.

Support media
A new DNA microarray device, S-Bio® PrimeSurface® for MPEX (BS-11608, Sumitomo Bakelite Co., Ltd, Tokyo, Japan), consists of COC grafted with an original biocompatible phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyehyleneglycol methacrylate (MEONP)] (PMBN) hydrophilic polymer (23). Such a biomembrane provides a highly active functional ester moiety to covalently bind the attachment site for C6-amino-oligonucleotides. PMBN was synthesized from the mole functions of each monomer unit (MPC, BMA and MEONP), which was about MPC/BMA/MEONP = 0.2/0.7/0.1. The activated PrimeSurface® slides were very stable for several months at room temperature and ~60% humidity (data not shown).

Atomic force microscopy
Atomic force microscopy (AFM, Dimension 3100, Veeco Instruments, USA) imaging experiments were operated in the tapping mode to obtain micrographs with high-resonance frequency for scanning the surface of the COC. Scan rates were set between 5 and 8 Hz depending on the image quality, and the scan size was changed from 1 to 10 µm upon engagement of the cantilever. All measurements were carried out under air-ambient conditions (temperature of 25°C and relative humidity of 60%).

Array design, preparation of synthetic oligonucleotides, spotting and DNA capture probe immobilization
The oligonucleotide probe set (Gene A) was designed to hybridize to an endogenous transcript present in mRNA from mouse cerebrum. The probe set Gene A consists of single-stranded 15, 20, 25, 30, 35 and 50mer 5'-C6-amino-oligonucleotides, which are the targeted transcripts of mice. These probe sequences are shown in Table 1. The target 5'-Cy5-labeled complementary 50mer synthesized oligonucleotide for Gene A: 5'-Cy5-AAGGCGGGAGGGAGCGCAATCCGGGAGTTTACAAATGGACAAACTTCTAT-3' was prepared for conventional hybridization and the MPEX template on the S-Bio® plastic slide. All DNA oligonucleotides were ordered and purchased from Fasmac (Tokyo, Japan). In order to immobilize these oligonucleotides onto the S-Bio® PrimeSurface® plastic slide, the 5' ends of oligonucleotides were modified with the C6 amino group. All oligonucleotides were desalted and purified by high-performance liquid chromatography (HPLC). The purity level of all oligonucleotides was systematically checked by HPLC before use.


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  		Table 1 Oligonucleotide sequences used in this study

 
The oligonucleotides were dissolved in 250 mM sodium carbonate buffer (pH 9.0), as the spotting solution, to a final concentration of 10 µM. 5'-Amine-terminated oligonucleotides were deposited onto the PrimeSurface® by mechanical spotting with a robot SPBIO® II (Hitachi Software Engineering Co., Tokyo, Japan). The spots were ca. 100 µm in diameter and set 300 µm apart. After administration of the nucleic acids, the slides were incubated overnight in a humid chamber with 250 mM sodium phosphate buffer at room temperature. The excess amine-reactive group (MEONP) was inactivated for 5 min at room temperature in blocking buffer solution containing 0.5 N NaOH. Finally, the DNA slides were washed in boiling water for 2 min, in water at room temperature for 2 min and then dried by centrifugation for 2 min at 200 g. The DNA arrays were then stored in a desiccated state at 4°C until use.

Hybridization to oligonucleotide DNA arrays
In all experiments, hybridization was carried out using our homemade gasket-type hybridization cassettes, with an inner volume of 50 µl. For oligonucleotide hybridization, a 500 pM solution of 5'-Cy5-labeled oligomer probe was dissolved in hybridization buffer (1x SSC, 0.5% SDS) under standard conditions. Usually, a portion of the 50 µl reaction mixture was used directly without further modification. The slides were pre-warmed for 45 min at 60°C, and hybridization was carried out in a chamber at 45°C or 60°C for 2 h in a 5'-Cy5-labeled complementary oligonucleotide target mixture. After hybridization, the slides were washed at the hybridization temperature, 45°C or 60°C, with the pre-made washing buffer 10x SSC with 0.5% (v/v) SDS, continuously in 1x SSC and then 0.1x SSC washing buffer for 1 min each and dried by centrifugation for 2 min at 200 g. After drying under a stream of dry air, fluorescence signals were captured with a laser scanner CRBIO® IIe (Hitachi Software Engineering Co., Tokyo, Japan) and the signal intensities were determined using DNASISArray® software (Hitachi Software Engineering Co., Ltd).

Terminal deoxynucleotidyl transferase reactions
The immobilized arrays were pre-heated to 37°C in a homemade hybridization chamber. A reaction mixture (150 µl) containing 10 µM Cy3-labeled dUTP (PerkinElmer), 2.0 U/µl terminal deoxynucleotidyl transferase (TdT, Roche), 3.13 mM CoCl2, 250 mM potassium cacodylate (pH 6.6), 31.3 mM Tris–HCl and 313 µg/ml BSA was overlaid onto the microarray slide surface. The extension reaction was allowed to proceed for 10 min at 37°C after which the slides were washed with dH2O in a solution of 1x SSC with 0.1% (v/v) SDS, and finally with dH2O.

Elimination of the hybridized oligonucleotide DNA targets
After immobilization, the capture probe at the 3' terminal was made to fluoresce using Cy3 through TdT reactions. The 5'-Cy5-labeled organic synthesized complementary target was hybridized at a concentration of 500 pM. To eliminate the oligonucleotide DNA target, the arrays were incubated in 0.1% (v/v) SDS water at 95°C for 1 min. The arrays were then washed with dH2O to remove SDS and dried for storage by centrifugation for 2 min at 200 g. Before and after each hybridization, the DNA arrays were checked by fluorescence scanning for 5'-Cy5-labeled target removal prior to the next hybridization and retainment of the immobilized 3'-Cy3-labeled probe DNA on the slide.

Primer extension using the synthetic oligonucleotide template
DNA amplification was initiated on the PrimeSurface® plastic slides with a primer extension reaction mixture containing 1 U/100 µl of EX Taq polymerase in 1x EX Taq buffer (TaKaRa Biosciences Co. Ltd, Otsu, Japan), 0.05 mM each of dATP, dCTP, dGTP (GibcoBRL®) and 0.05 mM Cy3-labeled dUTP, supplemented with 5'-Cy5-labeled target DNA mixture as a template from 0.1 to 1000 pM. Our homemade hybridization cassette was immersed in 50 µl of the reaction mixture in a frame seal chamber. Thermocycling was carried out as follows: 95°C for 5 min and 30 cycles (a denaturing step at 95°C for 1 min, an annealing step at 50°C for 3 min) using a GeneAmp® PCR System 9700 (Applied Biosystems). DNA arrays were washed with pre-prepared washing buffer as described previously and dried by centrifugation for 2 min at 200 g. After checking the fluorescence signals of the extended primers and the hybridized template DNA, the arrays were incubated in stripping 0.1% (v/v) SDS water at 95°C for 1 min, then washed with dH2O to remove SDS and dried by centrifugation for 2 min at 200 g.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND FUTURE...
 SUPPLEMENTARY DATA
 REFERENCES
 
Surface chemistry of S-Bio® PrimeSurface®
DNA array devices were prepared from a COC, which was used as an alternative of glass. COC has a strong reputation for having optical properties equivalent to PMMA (polymethyl methacrylate) as well as thermal resistance and dimensional stability superior to PMMA. COC also offers stiffness, very good compatibility with polyolefins and a high water vapor barrier. It is easy to fabricate, is inexpensive and has solvent resistance. It was compared with another polymer material, PMMA, which is also known as a good material candidate for bio-chips (24–26). As COC surfaces usually have strong hydrophobic interactions, they were not considered to be suitable for DNA–DNA hybridization and MPEX by DNA polymerase on a chip. To minimize these problems and improve COC performance for the preparation of DNA arrays, the surface was coated with a phospholipid polymer (PMBN) consisting of MPC, BMA and MEONP units. To the best of our knowledge, this is the first report of the use of COC for DNA chip devices to detect DNA–DNA hybridization properties.

In aqueous solution, BMA forms aggregates and becomes adsorbed onto a hydrophobic substrate surface, such as a COC surface (27). On the other hand, the hydrophilic MPC unit is oriented toward the aqueous solution. MEONP works as an active ester unit to conjugate with covalent binding though 5'-C6-amino-oligonucleotides as a DNA capture probe (primer) as shown in Figure 2A.


Figure 2
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  		Figure 2 (A) Chemical structure of PMBN. (B) AFM images of the COC surface with PMBN-coated surface structure. The mean roughness (Ra) was (a) 1.18 nm without coating and (b) 0.29 nm with PMBN.

 
The surface morphology of PMBN on the COC substrate was characterized using atomic force microscopy (AFM). Atomic force micrographs were taken at a 1.0 µm scan size and presented on a data scale of 10 nm for each of the surfaces studied as shown in Figure 2B. The mean roughness (Ra) of the COC plastic surface was smoothed on treatment by phospholipid polymerization (PMBN). This appears to be important for DNA microarrays to prevent the undesired non-specific adsorption of proteins, nucleotides, substrates such as dNTP and their labeled compounds. The efficiency of the MPEX reaction was compared for two different kinds of plastic DNA array devices: phospholipid polymer (PMBN)-coated plastic (S-Bio® PrimeSurface® for MPEX) and chemically aldehyde-modified slide (S-Bio® PrimeSurface® aldehyde) on the surface of the COC under MPEX reaction conditions (see Supplementary Figure 1).

Continuous enzyme reactions such as the MPEX reaction by DNA polymerase may occur on the surface of the plastic substrate S-Bio® PrimeSurface® (Figure 1). The characteristics and applications of this material are presented below.

Hybridization properties of the new plastic device
The sensitivity of duplex formation on PrimeSurface® was assayed as a function of the probe oligonucleotide length, demonstrated by comparing the hybridization of a complementary DNA target to an immobilized C6-amino-oligonucleotide probe (Gene A set) on the S-Bio® surface. Among the oligonucleotide probes ranging from 15 to 50mers and at two different hybridization temperatures, 45 and 60°C, the hybridization experiments were assessed as shown in Figure 3. Each oligonucleotide length of 25, 30, 35 and 50mers showed a signal-to-noise (S/N) ratio of over 100. The average hybridization sensitivity was highest at 50mer, decreasing slightly to 25mer at both temperatures. The oligonucleotide probes of 15 and 20mer were ineffective at 60°C. Notice that the calculated melting temperature (Tm) of the 20mer oligonucleotide sequence is roughly around 70°C.


Figure 3
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  		Figure 3 Comparison of hybridization signals obtained with different length oligonucleotides (15, 20, 25, 30, 35 and 50mer) and temperatures. The S-Bio® DNA array was hybridized to a 500 pM solution of Cy5 labeled oligo DNA target(5'-Cy5-AAGGCGGGAGGGAGCGCAATCCGGGAGTTTACAAATGGACAAACTTCTAT-3') complementary to the 50mer probe sequence.

 
To quantify hybridization sensitivity, a fixed amount of a 50mer oligonucleotide probe spotted as a 10 µM solution was hybridized for 16 h at 45°C with increasing concentrations of a 5'-Cy5-labeled complementary 50mer target from 0.1 to 1000 pM. As shown in Figure 4A, a plateau was reached at a target concentration of 500 pM. The lowest detection limit was around 1 pM where the signal-to-noise (S/N) ratio was still around 10, as shown in Figure 4B. A linear response was seen with a target concentration of 0.5–200 pM. The lowest detection sensitivity was ~10-fold higher than the aldehyde-modified plastic slide (S-Bio® PrimeSurface® aldehyde, BS-11101). The hybridization sensitivity of the plastic substrate S-Bio® PrimeSurface® was compared with a commercially available aldehyde-modified glass slide for DNA microarray (see Supplementary Figure 2). The result seems to be similar to that of the aldehyde-modified plastic slide.


Figure 4
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  		Figure 4 (A) Sensitivity and dynamic range: dependence of hybridization intensities on target concentration. Hybridizations were performed between the Cy5-labeled oligo DNA target (5'-Cy5-AAGGCGGGAGGGAGCGCAATCCGGGAGTTTACAAATGGACAAACTTCTAT-3') at concentrations ranging from 0.1 to 1000 pM and the complementary 50mer probe sequence. The fluorescence intensity reported is the fluorescent signal intensity. (B) The S/N ratios for hybridization signals were compared using two different products, PMBN-coated slide (S-Bio® PrimeSurface® for MPEX, BS-11608) and aldehyde-modified slide (S-Bio® PrimeSurface® aldehyde, BS-11101) which is an alternative to glass slides for producing hybridization-base DNA microarrays and oligonucleotide concentrations from 1 to 100 pM. Both of our products are made of cyclic olefin copolymer (COC).

 
To examine the specificity via mismatch on the PrimeSurface® surface, 25 and 30mer probes with a series of single-base and three-base or five-base mismatches in the middle of the oligonucleotide were designed and tested (see Supplementary Figure 3). These hybridization results seem to be similar to those of the CodeLinkTM platform (28,29), suggesting that the 30mer oligonucleotide probe may be accessible for ordinary hybridization with higher sensitivity and specificity in most, if not all, cases.

Thermal stability of the immobilized oligonucleotide DNA microarrays
To monitor the stability of the immobilized probe DNA on the PrimeSurface®, the capture probe at the 3' terminal was fluoresced using Cy3 via the terminal deoxynucleotidyl transferase (TdT) reaction. We tested whether the 50mer oligonucleotide probe could be hybridized with the complementary 50mer target, followed by stripping under dissociation conditions, and checked for any incompletely removed targets. The slide was then subjected to second cycle hybridization with the same target. The arrays manufactured on the PrimeSurface® showed no significant loss of hybridization activity for at least six cycles (data not shown). These results indicate that the probes are stably maintained on the plastic surface grafted with MPC and that this system could be subjected to repeated cycles, one of our important development aims.

In contrast to our results, the thermal stability of oligonucleotides bound to a conventionally modified glass surface is not robust, and in most cases, the retention of bound oligonucleotide DNA probes following denaturalization treatment is only ~50% (30,31). Adessi et al. (32) suggested that oligonucleotide release is not necessarily due to the stability of the covalent bond between the oligonucleotide and the cross-linker. They suspected that the cleavage of the bond between the cross-linker and the glass surface, as well as the potential instability of the glass surface itself, contributes to the release of bound oligonucleotides.

The possibility of reusing the oligonucleotide DNA microarray platform would be an advantage in gene transcription studies, as it would enable reproducibility of experiments on the same technical support. It would also reduce the cost of general two-color competitive hybridization experiments, while improving the accuracy of the genomic data. Thus, we investigated the reusability of oligonucleotide DNA microarray platforms generated from the PrimeSurface® plastic device. The same advantages were claimed with many other activated glass slides that allow covalent linkage of the probes (13,33,34); however, they did not allow reuse.

Multiple primer extension (MPEX) on S-BIO® by Taq DNA polymerase
As hybridization is reproducible on PrimeSurface® when the 5'-anchored oligonucleotide probe hybridizes with target DNA, the 3' terminus of the former can often be used for primer extension using the extra region of the target DNA as a primer (see Figure 1). We added 5'-terminus Cy5-labeled target DNA as a template in small amounts relative to the primer molecules, to allow primer extension in the presence of Cy3-dUTP and the denaturalization–annealing–extension cycles to proceed.

The amount of Cy3-labeled product by the primer extension reaction varied with the concentration of 100 pM of template DNA in solution and fixed using 30 cycles. Although the levels of fluorescence intensities were indeed dependent on the initial target DNA concentration and on the number of primer extension cycles, unexpectedly, the fluorescence intensities observed with 100 pM target DNA concentrated solutions were high for oligonucleotide DNA primers of all lengths, as shown in Figure 5. We also examined the effects of the probe length. The 15 and 20mer, as well as the 25, 30 and 35mer primers at a concentration of 100 pM almost reached the saturated signal intensity levels. These results indicate a detection sensitivity of the primer extension method on the PrimeSurface® of about an additional 10-fold higher than general hybridization signals and suggest that a much lower concentration of target DNA templates allowed detection at concentration ranges below the picomolar level (data not shown). We have demonstrated that when we used oligonucleotide DNA primers fixed on S-Bio® PrimeSurface®, and targeted a template in solution, the sequence-specific primer extension reaction took place on the solid surface under thermo cycling conditions. As shown in Figure 1, our new method can be used for DNA detection via MPEX by DNA polymerase. DNA templates hybridize to immobilized primers on the PMBN-coated slide, which are then elongated with DNA polymerase and dNTP as extender units incorporated into their nucleic acid chain elongation products. Our results also suggest that the DNA amplification procedure on the solid surface takes place via a mechanism similar to that of the PCR method.


Figure 5
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  		Figure 5 Comparison of intensity values resulting from MPEX reactions obtained on different length oligonucleotides (15, 20, 25, 30 and 35mer). The MPEX reactions on the S-Bio® DNA array were performed with a 100 pM solution of Cy5-labeled oligo DNA template (5'-Cy5-AAGGCGGGAGGGAGCGCAATCCGGGAGTTTACAAATGGACAAACTTCTAT-3') complementary to the 50mer probe sequence. The fluorescence intensity values for MPEX reaction (Primer Cy3 signals: red bars, Template Hybridization Cy5 signals: yellow bars) are plotted against the primer length, and other data (Primers Cy3 signals: blue bars, Template Hybridization Cy5 signals: light blue bars) are in the graph, which are the intensities after the stripped procedure with 0.1% SDS at 95°C for 1 min. The fluorescent signal intensities were captured with a laser scanner CRBIO® IIe and the signal intensities were determined with DNASISArray® software.

 
As far as we know, this study is the first to offer evidence of sequential target DNA amplification for a picomolar concentration range of the complementary oligonucleotide template. A similar proposal has appeared in the literature (32,35–43). Most studies focused on the solid-phase PCR method, which is a combination of PCR amplification on a chip and extension of the immobilized primer. Our approach offers a potentially useful technique for SNP typing and genome mutations. Problems remaining to be solved are loss of DNA primer during the primer extension cycles and interference between liquid-phase and solid-phase primers.

Kinetic study for MPEX
To understand the dynamics of MPEX on the S-Bio® PrimeSurface®, we examined the kinetic profile of MPEX under two different conditions, thermal cycling such as PCR and a constant temperature at 37°C. The data for the immobilized 20mer primer (Gene A) are plotted in Figure 6. The MPEX amplification fluorescence signals rose with increasing numbers of thermocycling at a concentration of 100 pM of template DNA solution in Figure 6A. Increasing the number of PCR cycles allowed detection of target DNA templates in the picomolar concentration range. This result indicates that the MPEX amplification reaction is indeed dependent on the number of amplification cycles as predicted by the interfacial linear amplification behavior model in Figure 1. The kinetics appearance of the MPEX reaction at 37°C was followed for 300 min in Figure 6B. MPEX amplification at 500 pM template DNA proceeded linearly on both surfaces during the first 30 to 120 min, approaching a steady-state between 150 and 300 min. Similar profiles were obtained for arrayed species of other different primers and templates (data not shown). In these experiments, both kinetics curves demonstrated similar linearity under two different MPEX conditions between thermal cycling such as PCR and a constant temperature at 37°C. Furthermore, signal intensity at any given time was proportional to the concentration of EX Taq DNA polymerase in the source surface, as shown in Figure 6C. The shape of this curve demonstrates that the reaction rates were dependent on the concentration of DNA polymerase in the source plastic plate at a picomolar concentration range of template DNA. This is the first evidence of MPEX amplification occurring on the surface of S-Bio® PrimeSurface® at a constant temperature. In addition, the DNA template, which is present in solution at a picomolar concentration level, can be highly amplified exclusively on a plastic plate grafted with a novel MPC polymer.


Figure 6
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  		Figure 6 Kinetics and efficiency of the MPEX reaction on S-Bio® PrimeSurface®. (A) Thermocycling procedure (a denaturing step at 95°C for 1 min, an annealing step at 50°C for 3 min) in the presence of a buffer solution of 100 pM template DNA, nucleotides (dNTPs) including with Cy3-labeled dUTP and Taq DNA polymerase resulted in DNA amplification from 1 to 20 cycles. (B) The kinetics time course of the MPEX amplification reaction in the concentration of 500 pM template DNA proceeded linearly on both surfaces during the first 30 to 120 min at a constant temperature of 37°C. (C) Effect of varying the Taq DNA polymerase concentration from 1U to 8U under the same conditions as (B).

 
Sequencing by synthesis (SBS) via MPEX biosynthesis
Sequencing By Hybridization (SBH) was developed in the late 1980s as an alternative to gel-based sequencing. In general, it uses a universal DNA microarray, which harbors all probe oligonucleotide sets, e.g. 5–10mer on a membrane or a glass chip. These oligonucleotide probes are hybridized to an unknown DNA fragment, the sequence of which remains to be determined (10,44–48).

We have developed Sequencing By Synthesis (SBS) via MPEX biosynthesis such as SBH examined the detection limit of oligonucleotide lengths of less than 15mer. The results with 30 cycles of primer extension reaction using relatively shorter length oligonucleotide primers sets were obtained at a concentration of 500 pM DNA template, as shown in Figure 7. Laser scanning image resulting from MPEX reaction led to different lengths of oligonucleotides (8, 10 and 15mer) and DNA sequences to the complementary template DNA. The primer extension signal of DNA sequence No. 7 (10mer) and No. 13 (8mer), which have lower GC% in these sequences as shown in Figure 7, considerably decreased corresponding to other 8 and 10mer primers. These results suggested that the shorter-length oligonucleotides such as 8mer could be discriminated by the template DNA via the MPEX biosynthesis. Further work is necessary to validate this approach with actual data.


Figure 7
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  		Figure 7 Laser scanning image resulting from MPEX reaction obtained on different short length oligonucleotides (8, 10 and 15mer) and DNA sequences via complementary template (5'-Cy5-AAGGCGGGAGGGAGCGCAATCCGGGAGTTTACAAATGGACAAACTTCTAT-3'). Thermocycling procedure (a denaturing step at 95°C for 1 min, an annealing step at 30°C for 5 min) in the presence of a buffer solution of 500 pM template DNA, nucleotides (dNTPs) including with Cy3-labeled dUTP and Taq DNA polymerase resulted on 30 cycles of DNA amplification.

 

   CONCLUSIONS AND FUTURE CHALLENGES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND FUTURE...
SUPPLEMENTARY DATA
 REFERENCES
 
We have demonstrated that DNA oligonucleotide probes are covalently immobilized on the PrimeSurface® plastic surface via an amine at their 5' terminus. This type of surface chemistry offers extraordinarily stable thermal properties because of the absence of a pre-activated glass slide surface. Our results suggest that the plastic platform S-BIO® PrimeSurface® can clear challenging technical hurdles for DNA microarrays in hybridization–based analysis in the near future. In addition, when the oligonucleotide DNA template is in solution on DNA arrays, the sequence-specific primer extension reaction and sequential DNA amplification can be processed on the solid surface by thermal cycling such as used in the PCR method. In this case, oligonucleotide DNA primers on the PrimeSurface® can be highly amplified exclusively at the picomolar concentration range of complementary oligonucleotide templates. The major advantage of the MPEX method over the hybridization method is that a single set of optimal reaction conditions can be used to genotype all sequence variants, enabling simplification and optimization of the assay design. Finally, we have demonstrated that the MPEX reaction condition can be simplified as shown by the kinetics study at a constant temperature. These results suggested that totally different approaches of genome analysis such as SNP analysis, SBS via MPEX biosynthesis and the detection of noncording micro RNA by reverse transcriptase can be developed using our DNA microarray platform PrimeSurface® through MPEX techniques. Further work on device development is needed to enable precision control of the temperature because the plastic substrate generally has lower thermoconductivity than a glass slide.

We conclude by stressing that while hybridization–based sequencing assays are still in their infancy, improvement of our primer extension method should offer an even more powerful analytical tool for accurate and high–throughput resequencing strategy and mutational analysis. We will focus on simplifying the analytical procedure, such as of the DNA polymerase reaction conditions and preparation chemistry, to extend the technology for the analysis of nucleic acids (DNA and RNA) extracted from clinical and environmental samples. Our method using a PMBN-coated plastic substrate has the potential to become a widely applicable tool for laboratories performing large-scale analyses and for use as a DNA microarray platform.


   SUPPLEMENTARY DATA
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND FUTURE...
 SUPPLEMENTARY DATA
REFERENCES
 
Supplementary Data are available at NAR Online.


   ACKNOWLEDGEMENTS
 
The authors thank Professor Hidetoshi Inoko (Tokai University, School of Medicine), Dr Takashi Imai and Dr Yuichi Michikawa (National Institute of Radiological Sciences) for their helpful suggestions and discussions. Funding to pay the Open Access publication charges for this article was provided by Ministry of Health, Labor and Welfare in Japan.

Conflict of interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND FUTURE...
 SUPPLEMENTARY DATA
 REFERENCES
 

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