- Email: [email protected]
Whole genome amplification on poly(dimethylsiloxane) microchip array
Available online at www.sciencedirect.com
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 372 (2008) 128–130 www.elsevier.com/locate/yabio
Notes & Tips
Whole genome amplification on poly(dimethylsiloxane) microchip array Lin Chen a, Andreas Manz a, Philip J.R. Day b
a,b,*
a Institute for Analytical Sciences, Bunsen-Kirchhoff Str. 11, D-44139 Dortmund, Germany The Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK
Received 19 June 2007 Available online 1 September 2007
The availability of large quantities of genomic DNA possessing a suitable quality for nucleic acid analyses is of critical importance for many applications such as highthroughput genotyping assays, forensic science, embryonic disease diagnosis, and, importantly, long-term DNA sample storage including national and disease-specific DNA archives [1]. However, often the amount of available DNA is insufficient for extensive analyses. This situation is more challenging with the analysis of single cells where only finite testing can be undertaken. To address this problem, several methods for whole genome amplification (WGA)1 have been developed over the past 10 years, including degenerate oligonucleotide primed polymerase chain reaction, primer extension preamplification, and multiple displacement amplification (MDA). Compared to the other WGA methods, MDA appears to be the most reliable for genotyping since it has the better genomic coverage, the lowest sequence-related amplification bias, and the most favorable call rates [2–4]. Moreover, MDA is an isothermal amplification process, which avoids the need for thermocycling. It has been applied to amplify DNA from single bacterial cells to allow genome sequencing [5,6]. The application of lab-on-a-chip devices in molecular biology has bloomed in recent years due to distinct advantages of higher sensitivity, lower reagent consumption, and integration of different functional units [7,8]. We show in this study that MDA-based WGA can be readily adapted to miniaturized systems, maintains the possibility of providing a sustainable DNA archive compared to currently employed procedures, and with integration may offer a contamination-free and more cost-effective means to provide a preamplification unit for subsequent genotyping. *
Corresponding author. Fax.: +49 231 1392 120. E-mail address: [email protected] (P.J.R. Day). 1 Abbreviations used: WGA, Whole genome amplification; MDA, multiple displacement amplification; PDMS, poly(dimethylsiloxane); NTC, no template control; SNP, single-nucleotide polymorphism. 0003-2697/$ - see front matter 2007 Published by Elsevier Inc. doi:10.1016/j.ab.2007.07.036
The aluminum master mould for microchip fabrication was constructed using micromachining. The master contained 70 cylinders (2 mm in both diameter and depth) with 4 mm distance between cylinder centers. Poly(dimethylsiloxane) (PDMS) prepolymer and curing reagent (Sylgard 184) were thoroughly mixed, degassed, and then poured onto the aluminum master mould. After curing at 80 C for 1 h, the PDMS was peeled off from the master and bound to a glass slide (60 · 24 · 0.1 mm) after oxygen plasma treatment (shown in Fig. 1B). For silanization, the whole PDMS microchip was rinsed with ethanol and pure water and dried under hydrogen gas. Then selected wells were washed with acetone and chloroform, filled with silanization solution (5% dicholorodimethylsilane in chloroform), incubated for 30 min, and dried under hydrogen gas. Genomiphi (GE Healthcare) sample buffer (9 lL) and human genomic DNA (1 lL) were mixed on ice, and then GenomiPhi reaction buffer (9 lL) and Phi29 polymerase (1 lL) were added. For both conventional and on-chip real-time WGA monitoring, additional 0.2· SYBR I (Invitrogen) was added. After gently vortexing and centrifuging, the samples were incubated at 30 C for 90 min and then 65 C for 10 min (to inactivate the Phi29 polymerase) using a conventional thermocycler (Mastercycler 5332, Eppendorf). For on-chip WGA, 2 lL of sample prepared as above was pipetted and loaded into a well on a PDMS microchip. To prevent the evaporation of the reaction mixture, mineral oil was used to cover the reaction well. The whole chip was incubated at 30 C for 90 min, followed by 65 C for 10 min. Conventional WGA real-time monitoring was carried out on ABI PRISM HT7900. Fluorescence intensities were collected via the SYBR green I channel. For on-chip real-time WGA monitoring, the chip was placed on the top of two Peltier elements mounted on a microscope stage. The temperature of the wells for analysis was maintained at 30 C and fluorescence intensity was measured approx every 1 min by manually opening and
Notes & Tips / Anal. Biochem. 372 (2008) 128–130
Fig. 1. (A) Gel electrophoresis analysis of WGA products. Lane L, 1-kb DNA ladder; lanes 1–4, WGA products amplified from the conventional thermocycler with different initial amounts of human genomic DNA. Lane 1, 10 ng; lane 2, 1 ng; lane 3, 100 pg; lane 4, 10 pg; lane 5, conventional WGA NTC; lanes 6 and 7, WGA products amplified from silanized (lane 6) and native (lane 7) PDMS microchip with an initial human genomic DNA amount of 1 ng; lane 8, NTC WGA product from microchip; lane 9, unamplified human genomic DNA. (B) Photograph of 70-well PDMS microchip array used for on-chip WGA.
closing the shutter to the photo multiplier tube. For all conventional and on-chip WGA experiments, no-template control (NTC) for each sample is run in parallel by replacing DNA template with 1 lL water. Both amplified and unamplified human genomic DNA samples were used as templates for subsequent real-time PCR using the ABI TaqMan RNase P detection reagent kit; 1 lL of unamplified genomic DNA (1 ng), 1 lL of conventional WGA product, and 1 lL of on-chip WGA product (both amplified from 1 ng human genomic DNA) were used as templates, respectively. Loci representation efficiency (WGA product/unamplified DNA) is reported as (loci copy number/mass of WGA products)/(loci copy number/mass of unamplified DNA) [3,9]. Although the protocol from the manufacturer recommended an initial denaturation step at 95 C for the sample mixture, from our study, without this step, the results were routinely seen to at least resemble the procedure employing denaturation (data not shown) and is in agreement with other reports [2,9]. The noninclusion of the initial denaturation is advantageous for the temperature programming of the
129
microchip-based MDA. Thus we perform both our conventional and on-chip MDA without the initial denature step. Due to surface chemistry, PDMS and glass devices suffer from adsorption of DNA and enzymes, which is a generally accepted observation in microchip-based PCR. Silanization is a well-established surface modification method to improve DNA amplification efficiency. In our experiments, we observed that the amount of WGA products from a native PDMS well is less than half of that from a silanized PDMS well, which suggests that silanization is a necessary step to enhance MDA efficiency. Silanized microchips were used throughout the experiments. The on-chip MDA generated DNA products of 10–40 kb, similar to that of conventional amplified WGA and unamplified genomic DNA, as determined by 0.8% agarose gel electrophoresis (Fig. 1A). High-molecular-weight amplified DNA products are ideal for DNA library construction and enable genomic sequencing from one or a few cells, in contrast to greater bias in sequence representation of the WGA products obtained using other PCR-based methods where products are only a few hundred basepairs. The MDA has an exponential stage like PCR, and the time it takes to measurably reach this geometric phase is related to the initial template concentration [6]. As can be seen from Fig. 2, the on-chip real-time MDA detection with different initial amounts of DNA was in a good agreement with that of those performed on a conventional larger-volume machine. Note too, as with conventional use of gene amplification including PCR, the higher concentration of template can cause inhibition to overall amplified products. The utility of MDA-based WGA product as enriched template for further downstream applications such as real-time PCR was investigated. Compared to unamplified human genomic DNA, the Ct value of chip WGA product is reduced by 8.0, which corresponds to more than 250fold increase in initial DNA template amount when used as the templates for real-time quantitative PCR detection of RNase P (Fig. 2C). Compared to the amplification factor (400-fold) achieved with conventional WGA, the slightly lower amplification factor (250-fold) of on-chip WGA is probably due to the limited reagents (dNTPs and primers)
Fig. 2. (A) Real-time detection of MDA on conventional real-time PCR machine (ABI PRISM HT 7900); (B) real-time detection of MDA on PDMS microchip; (C) real-time PCR detection of RNase P using 1 lL unamplified genomic DNA (1 ng), 1 lL conventional WGA product, and 1 lL chip WGA product, both amplified from 1 ng human genomic DNA, respectively. NTC for each sample is run in parallel.
130
Notes & Tips / Anal. Biochem. 372 (2008) 128–130
in the 10-fold lower reaction volume, although both started with the same amount of genomic DNA. The specific DNA yields obtained by real-time PCR (chip WGA: 241.6 ± 3.0 ng/lL, n = 3; conventional WGA: 428.6 ± 5.4 ng/lL, n = 3) and total DNA concentration measured by Nanodrop spectrometer (ND-1000) (chip WGA: 252.1 ± 3.5 ng/lL, n = 3; conventional WGA: 522.0 ± 10.5 ng/lL, n = 3) reflect representation efficiencies of 95.8% for on-chip WGA and 82.1% for conventional WGA. The higher representation efficiency of on-chip WGA product is probably due to the reduced reaction volume and the relative high ratio of template in the reaction mixture, although this is not proven in the reported studies [5,10]. For WGA, the fidelity associated with amplicon synthesis is a critical issue because WGA products must perform similarly to the unamplified genomic sample in subsequent genetic analysis, particularly single-nucleotide polymorphism (SNP) genotyping. We used TaqMan SNP Genotyping Assays to test on-chip WGA products and unamplified genomic DNA from eight individuals for two SNPs (SNP1: rs2069718, A/G; SNP2: rs13900, C/T). In all eight cases, onchip WGA products were correctly genotyped as compared with results from larger samples not subjected to WGA. It is worthy to note that 54 samples were subjected to WGA (six aliquots for each of the eight samples and negative control) and simultaneously run on the same chip, indicating that the system may offer a basis for a high-throughput PCR preamplification platform for large-scale SNP scoring. More SNP analysis testing is being carried out to assess the fidelity of on-chip WGA products. In all our experiments for on-chip NTC WGA products, we have not observed any background synthesis and all SNP analyses showed the correct genotype calling. This is probably due to the reduced reaction volume and increased ratio of template in relation to any reaction components that are responsible or contribute to background [5,10]. Therefore, the higher concentration of on-chip-amplified DNA (see Fig. 1) and higher representation efficiency of 95.8% compared to 82.1% of conventional WGA can be obtained on miniaturized platforms. This could be a potential advantage of microchip-based MDA for amplifying very small amounts of DNA, because the background synthesis has become a critical issue for MDA reactions. These studies reveal for the first time the use of a microchip device for multiple displacement amplification WGA. The RNase P assay indicated that the level of amplification is close to 250-fold compared to the initial input template genomic DNA when using 2 lL reaction volume on PDMS microchips. The high representation efficiency of 95.8% reflects a high specificity of the on-chip MDA reaction for amplifying genomic sequence. The fidelity of the PCR appears to be extremely high as suggested by the perfect analysis score of the SNPs analyzed which reflects the actual sequences unveiled by conventional SNP-based PCRs. Our experiments showed that commercial WGA reagents are readily adapted to a microchip platform without further modification. Although the 2-lL reaction volume of our
on-chip WGA is slightly higher than the reported 0.5-lL reaction volume of a 384-well microplate using a special sample loading device [11], we believe that the benefits of lower sample and reagent consumption for miniaturized WGA can be better explored when coupling with a sample pretreatment, PCR, and sample analysis unit which are well studied in l-TAS, where general reaction volume is in the range of nL to pL (e.g., droplet reaction inside microfluidic devices). Therefore a 2-lL reaction is readily amenable to miniaturized packaging as shown here and permits huge numbers of ensuing tests to be carried out in integrated devices employing, for example, PCR analysis of SNPs. Moreover, unlike many reports of microfluidic devices in applications of PCR, where complex thermal control systems are required and only selected regions of DNA samples can be amplified, the use of WGA on-chip has many additional benefits. These relate to the simplified thermal requirement for DNA amplification, the tracking of the reaction, the long-term storage, and the perpetuation of important patient population cohorts within microfluidic device-based archives. Acknowledgments We thank Fiona Salway and Francine Jury for gel electrophoresis analysis. References [1] L. Lovmar, A.C. Syvanen, Multiple displacement amplification to create a long-lasting source of DNA for genetic studies, Hum. Mutat. 27 (2006) 603–614. [2] F.B. Dean, S. Hosono, L. Fang, X. Wu, A.F. Faruqi, P. Bray-Ward, Z. Sun, Q. Zong, Y. Du, J. Du, M. Driscoll, W. Song, S.F. Kingsmore, M. Egholm, R.S. Lasken, Comprehensive human genome amplification using multiple displacement amplification, Proc. Natl. Acad. Sci. USA 99 (2002) 5261–5266. [3] S. Hosono, A.F. Faruqi, F.B. Dean, Y. Du, Z. Sun, X. Wu, J. Du, S.F. Kingsmore, M. Egholm, R.S. Lasken, Unbiased whole-genome amplification directly from clinical samples, Genome Res. 13 (2003) 954–964. [4] J.W. Park, T.H. Beaty, P. Boyce, A.F. Scott, I. McIntosh, Comparing Whole-Genome Amplification Methods and Sources of Biological Samples for Single-Nucleotide Polymorphism Genotyping, Clin. Chem. 51 (2005) 1520–1523. [5] C.A. Hutchison 3rd., J.C. Venter, Single-cell genomics, Nat Biotechnol 24 (2006) 657–658. [6] K. Zhang, A.C. Martiny, N.B. Reppas, K.W. Barry, J. Malek, S.W. Chisholm, G.M. Church, Sequencing genomes from single cells by polymerase cloning, Nat. Biotechnol. 24 (2006) 680–686. [7] P.A. Auroux, Y. Koc, A. deMello, A. Manz, P.J. Day, Miniaturised nucleic acid analysis, Lab Chip 4 (2004) 534–546. [8] P.S. Dittrich, K. Tachikawa, A. Manz, Micro total analysis systems. Latest advancements and trends, Anal. Chem. 78 (2006) 3887–3908. [9] C. Spits, C. Le Caignec, M. De Rycke, L. Van Haute, A. Van Steirteghem, I. Liebaers, K. Sermon, Optimization and evaluation of single-cell whole-genome multiple displacement amplification, Hum. Mutat. 27 (2006) 496–503. [10] C.A. Hutchison 3rd., H.O. Smith, C. Pfannkoch, J.C. Venter, Cellfree cloning using phi29 DNA polymerase, Proc. Natl. Acad. Sci USA 102 (2005) 17332–17336. [11] H.C. Wu, J. Shieh, D.J. Wright, A. Azarani, DNA sequencing using rolling circle amplification and precision glass syringes in a high-throughput liquid handling system, Biotechniques 34 (2003) 204–207.
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 372 (2008) 128–130 www.elsevier.com/locate/yabio
Notes & Tips
Whole genome amplification on poly(dimethylsiloxane) microchip array Lin Chen a, Andreas Manz a, Philip J.R. Day b
a,b,*
a Institute for Analytical Sciences, Bunsen-Kirchhoff Str. 11, D-44139 Dortmund, Germany The Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK
Received 19 June 2007 Available online 1 September 2007
The availability of large quantities of genomic DNA possessing a suitable quality for nucleic acid analyses is of critical importance for many applications such as highthroughput genotyping assays, forensic science, embryonic disease diagnosis, and, importantly, long-term DNA sample storage including national and disease-specific DNA archives [1]. However, often the amount of available DNA is insufficient for extensive analyses. This situation is more challenging with the analysis of single cells where only finite testing can be undertaken. To address this problem, several methods for whole genome amplification (WGA)1 have been developed over the past 10 years, including degenerate oligonucleotide primed polymerase chain reaction, primer extension preamplification, and multiple displacement amplification (MDA). Compared to the other WGA methods, MDA appears to be the most reliable for genotyping since it has the better genomic coverage, the lowest sequence-related amplification bias, and the most favorable call rates [2–4]. Moreover, MDA is an isothermal amplification process, which avoids the need for thermocycling. It has been applied to amplify DNA from single bacterial cells to allow genome sequencing [5,6]. The application of lab-on-a-chip devices in molecular biology has bloomed in recent years due to distinct advantages of higher sensitivity, lower reagent consumption, and integration of different functional units [7,8]. We show in this study that MDA-based WGA can be readily adapted to miniaturized systems, maintains the possibility of providing a sustainable DNA archive compared to currently employed procedures, and with integration may offer a contamination-free and more cost-effective means to provide a preamplification unit for subsequent genotyping. *
Corresponding author. Fax.: +49 231 1392 120. E-mail address: [email protected] (P.J.R. Day). 1 Abbreviations used: WGA, Whole genome amplification; MDA, multiple displacement amplification; PDMS, poly(dimethylsiloxane); NTC, no template control; SNP, single-nucleotide polymorphism. 0003-2697/$ - see front matter 2007 Published by Elsevier Inc. doi:10.1016/j.ab.2007.07.036
The aluminum master mould for microchip fabrication was constructed using micromachining. The master contained 70 cylinders (2 mm in both diameter and depth) with 4 mm distance between cylinder centers. Poly(dimethylsiloxane) (PDMS) prepolymer and curing reagent (Sylgard 184) were thoroughly mixed, degassed, and then poured onto the aluminum master mould. After curing at 80 C for 1 h, the PDMS was peeled off from the master and bound to a glass slide (60 · 24 · 0.1 mm) after oxygen plasma treatment (shown in Fig. 1B). For silanization, the whole PDMS microchip was rinsed with ethanol and pure water and dried under hydrogen gas. Then selected wells were washed with acetone and chloroform, filled with silanization solution (5% dicholorodimethylsilane in chloroform), incubated for 30 min, and dried under hydrogen gas. Genomiphi (GE Healthcare) sample buffer (9 lL) and human genomic DNA (1 lL) were mixed on ice, and then GenomiPhi reaction buffer (9 lL) and Phi29 polymerase (1 lL) were added. For both conventional and on-chip real-time WGA monitoring, additional 0.2· SYBR I (Invitrogen) was added. After gently vortexing and centrifuging, the samples were incubated at 30 C for 90 min and then 65 C for 10 min (to inactivate the Phi29 polymerase) using a conventional thermocycler (Mastercycler 5332, Eppendorf). For on-chip WGA, 2 lL of sample prepared as above was pipetted and loaded into a well on a PDMS microchip. To prevent the evaporation of the reaction mixture, mineral oil was used to cover the reaction well. The whole chip was incubated at 30 C for 90 min, followed by 65 C for 10 min. Conventional WGA real-time monitoring was carried out on ABI PRISM HT7900. Fluorescence intensities were collected via the SYBR green I channel. For on-chip real-time WGA monitoring, the chip was placed on the top of two Peltier elements mounted on a microscope stage. The temperature of the wells for analysis was maintained at 30 C and fluorescence intensity was measured approx every 1 min by manually opening and
Notes & Tips / Anal. Biochem. 372 (2008) 128–130
Fig. 1. (A) Gel electrophoresis analysis of WGA products. Lane L, 1-kb DNA ladder; lanes 1–4, WGA products amplified from the conventional thermocycler with different initial amounts of human genomic DNA. Lane 1, 10 ng; lane 2, 1 ng; lane 3, 100 pg; lane 4, 10 pg; lane 5, conventional WGA NTC; lanes 6 and 7, WGA products amplified from silanized (lane 6) and native (lane 7) PDMS microchip with an initial human genomic DNA amount of 1 ng; lane 8, NTC WGA product from microchip; lane 9, unamplified human genomic DNA. (B) Photograph of 70-well PDMS microchip array used for on-chip WGA.
closing the shutter to the photo multiplier tube. For all conventional and on-chip WGA experiments, no-template control (NTC) for each sample is run in parallel by replacing DNA template with 1 lL water. Both amplified and unamplified human genomic DNA samples were used as templates for subsequent real-time PCR using the ABI TaqMan RNase P detection reagent kit; 1 lL of unamplified genomic DNA (1 ng), 1 lL of conventional WGA product, and 1 lL of on-chip WGA product (both amplified from 1 ng human genomic DNA) were used as templates, respectively. Loci representation efficiency (WGA product/unamplified DNA) is reported as (loci copy number/mass of WGA products)/(loci copy number/mass of unamplified DNA) [3,9]. Although the protocol from the manufacturer recommended an initial denaturation step at 95 C for the sample mixture, from our study, without this step, the results were routinely seen to at least resemble the procedure employing denaturation (data not shown) and is in agreement with other reports [2,9]. The noninclusion of the initial denaturation is advantageous for the temperature programming of the
129
microchip-based MDA. Thus we perform both our conventional and on-chip MDA without the initial denature step. Due to surface chemistry, PDMS and glass devices suffer from adsorption of DNA and enzymes, which is a generally accepted observation in microchip-based PCR. Silanization is a well-established surface modification method to improve DNA amplification efficiency. In our experiments, we observed that the amount of WGA products from a native PDMS well is less than half of that from a silanized PDMS well, which suggests that silanization is a necessary step to enhance MDA efficiency. Silanized microchips were used throughout the experiments. The on-chip MDA generated DNA products of 10–40 kb, similar to that of conventional amplified WGA and unamplified genomic DNA, as determined by 0.8% agarose gel electrophoresis (Fig. 1A). High-molecular-weight amplified DNA products are ideal for DNA library construction and enable genomic sequencing from one or a few cells, in contrast to greater bias in sequence representation of the WGA products obtained using other PCR-based methods where products are only a few hundred basepairs. The MDA has an exponential stage like PCR, and the time it takes to measurably reach this geometric phase is related to the initial template concentration [6]. As can be seen from Fig. 2, the on-chip real-time MDA detection with different initial amounts of DNA was in a good agreement with that of those performed on a conventional larger-volume machine. Note too, as with conventional use of gene amplification including PCR, the higher concentration of template can cause inhibition to overall amplified products. The utility of MDA-based WGA product as enriched template for further downstream applications such as real-time PCR was investigated. Compared to unamplified human genomic DNA, the Ct value of chip WGA product is reduced by 8.0, which corresponds to more than 250fold increase in initial DNA template amount when used as the templates for real-time quantitative PCR detection of RNase P (Fig. 2C). Compared to the amplification factor (400-fold) achieved with conventional WGA, the slightly lower amplification factor (250-fold) of on-chip WGA is probably due to the limited reagents (dNTPs and primers)
Fig. 2. (A) Real-time detection of MDA on conventional real-time PCR machine (ABI PRISM HT 7900); (B) real-time detection of MDA on PDMS microchip; (C) real-time PCR detection of RNase P using 1 lL unamplified genomic DNA (1 ng), 1 lL conventional WGA product, and 1 lL chip WGA product, both amplified from 1 ng human genomic DNA, respectively. NTC for each sample is run in parallel.
130
Notes & Tips / Anal. Biochem. 372 (2008) 128–130
in the 10-fold lower reaction volume, although both started with the same amount of genomic DNA. The specific DNA yields obtained by real-time PCR (chip WGA: 241.6 ± 3.0 ng/lL, n = 3; conventional WGA: 428.6 ± 5.4 ng/lL, n = 3) and total DNA concentration measured by Nanodrop spectrometer (ND-1000) (chip WGA: 252.1 ± 3.5 ng/lL, n = 3; conventional WGA: 522.0 ± 10.5 ng/lL, n = 3) reflect representation efficiencies of 95.8% for on-chip WGA and 82.1% for conventional WGA. The higher representation efficiency of on-chip WGA product is probably due to the reduced reaction volume and the relative high ratio of template in the reaction mixture, although this is not proven in the reported studies [5,10]. For WGA, the fidelity associated with amplicon synthesis is a critical issue because WGA products must perform similarly to the unamplified genomic sample in subsequent genetic analysis, particularly single-nucleotide polymorphism (SNP) genotyping. We used TaqMan SNP Genotyping Assays to test on-chip WGA products and unamplified genomic DNA from eight individuals for two SNPs (SNP1: rs2069718, A/G; SNP2: rs13900, C/T). In all eight cases, onchip WGA products were correctly genotyped as compared with results from larger samples not subjected to WGA. It is worthy to note that 54 samples were subjected to WGA (six aliquots for each of the eight samples and negative control) and simultaneously run on the same chip, indicating that the system may offer a basis for a high-throughput PCR preamplification platform for large-scale SNP scoring. More SNP analysis testing is being carried out to assess the fidelity of on-chip WGA products. In all our experiments for on-chip NTC WGA products, we have not observed any background synthesis and all SNP analyses showed the correct genotype calling. This is probably due to the reduced reaction volume and increased ratio of template in relation to any reaction components that are responsible or contribute to background [5,10]. Therefore, the higher concentration of on-chip-amplified DNA (see Fig. 1) and higher representation efficiency of 95.8% compared to 82.1% of conventional WGA can be obtained on miniaturized platforms. This could be a potential advantage of microchip-based MDA for amplifying very small amounts of DNA, because the background synthesis has become a critical issue for MDA reactions. These studies reveal for the first time the use of a microchip device for multiple displacement amplification WGA. The RNase P assay indicated that the level of amplification is close to 250-fold compared to the initial input template genomic DNA when using 2 lL reaction volume on PDMS microchips. The high representation efficiency of 95.8% reflects a high specificity of the on-chip MDA reaction for amplifying genomic sequence. The fidelity of the PCR appears to be extremely high as suggested by the perfect analysis score of the SNPs analyzed which reflects the actual sequences unveiled by conventional SNP-based PCRs. Our experiments showed that commercial WGA reagents are readily adapted to a microchip platform without further modification. Although the 2-lL reaction volume of our
on-chip WGA is slightly higher than the reported 0.5-lL reaction volume of a 384-well microplate using a special sample loading device [11], we believe that the benefits of lower sample and reagent consumption for miniaturized WGA can be better explored when coupling with a sample pretreatment, PCR, and sample analysis unit which are well studied in l-TAS, where general reaction volume is in the range of nL to pL (e.g., droplet reaction inside microfluidic devices). Therefore a 2-lL reaction is readily amenable to miniaturized packaging as shown here and permits huge numbers of ensuing tests to be carried out in integrated devices employing, for example, PCR analysis of SNPs. Moreover, unlike many reports of microfluidic devices in applications of PCR, where complex thermal control systems are required and only selected regions of DNA samples can be amplified, the use of WGA on-chip has many additional benefits. These relate to the simplified thermal requirement for DNA amplification, the tracking of the reaction, the long-term storage, and the perpetuation of important patient population cohorts within microfluidic device-based archives. Acknowledgments We thank Fiona Salway and Francine Jury for gel electrophoresis analysis. References [1] L. Lovmar, A.C. Syvanen, Multiple displacement amplification to create a long-lasting source of DNA for genetic studies, Hum. Mutat. 27 (2006) 603–614. [2] F.B. Dean, S. Hosono, L. Fang, X. Wu, A.F. Faruqi, P. Bray-Ward, Z. Sun, Q. Zong, Y. Du, J. Du, M. Driscoll, W. Song, S.F. Kingsmore, M. Egholm, R.S. Lasken, Comprehensive human genome amplification using multiple displacement amplification, Proc. Natl. Acad. Sci. USA 99 (2002) 5261–5266. [3] S. Hosono, A.F. Faruqi, F.B. Dean, Y. Du, Z. Sun, X. Wu, J. Du, S.F. Kingsmore, M. Egholm, R.S. Lasken, Unbiased whole-genome amplification directly from clinical samples, Genome Res. 13 (2003) 954–964. [4] J.W. Park, T.H. Beaty, P. Boyce, A.F. Scott, I. McIntosh, Comparing Whole-Genome Amplification Methods and Sources of Biological Samples for Single-Nucleotide Polymorphism Genotyping, Clin. Chem. 51 (2005) 1520–1523. [5] C.A. Hutchison 3rd., J.C. Venter, Single-cell genomics, Nat Biotechnol 24 (2006) 657–658. [6] K. Zhang, A.C. Martiny, N.B. Reppas, K.W. Barry, J. Malek, S.W. Chisholm, G.M. Church, Sequencing genomes from single cells by polymerase cloning, Nat. Biotechnol. 24 (2006) 680–686. [7] P.A. Auroux, Y. Koc, A. deMello, A. Manz, P.J. Day, Miniaturised nucleic acid analysis, Lab Chip 4 (2004) 534–546. [8] P.S. Dittrich, K. Tachikawa, A. Manz, Micro total analysis systems. Latest advancements and trends, Anal. Chem. 78 (2006) 3887–3908. [9] C. Spits, C. Le Caignec, M. De Rycke, L. Van Haute, A. Van Steirteghem, I. Liebaers, K. Sermon, Optimization and evaluation of single-cell whole-genome multiple displacement amplification, Hum. Mutat. 27 (2006) 496–503. [10] C.A. Hutchison 3rd., H.O. Smith, C. Pfannkoch, J.C. Venter, Cellfree cloning using phi29 DNA polymerase, Proc. Natl. Acad. Sci USA 102 (2005) 17332–17336. [11] H.C. Wu, J. Shieh, D.J. Wright, A. Azarani, DNA sequencing using rolling circle amplification and precision glass syringes in a high-throughput liquid handling system, Biotechniques 34 (2003) 204–207.