Scoring Single-Nucleotide Polymorphisms at the Single-Molecule

ability of RCA to produce single-stranded DNA tens of thousands of nucleotides in length from a single cleaved. DNA molecule on the surface suggested ...
0 downloads 0 Views 328KB Size
Download PDF
Anal. Chem. 2005, 77, 6594-6600

Scoring Single-Nucleotide Polymorphisms at the Single-Molecule Level by Counting Individual DNA Cleavage Events on Surfaces Bei Nie, Michael R. Shortreed, and Lloyd M. Smith*

Department of Chemistry, University of WisconsinsMadison, 1101 University Avenue, Madison, Wisconsin 53706

Single-nucleotide polymorphisms (SNPs) are the most frequent type of human genetic variation. Recent work has shown that it is possible to directly analyze SNPs in unamplified human genomic DNA samples using the surface-invasive cleavage reaction followed by rolling circle amplification (RCA) of the cleavage products. The ability of RCA to produce single-stranded DNA tens of thousands of nucleotides in length from a single cleaved DNA molecule on the surface suggested the possibility of detecting individual cleavage events on the surface. The feasibility of this approach to SNP scoring is shown here. Individual cleavage events on the surface are detected using fluorescence microscopy to visualize the singlestranded DNA product of the RCA reaction labeled with the fluorescent dye SYBR Green I. The surface density of fluorescent features observed is dependent upon the concentration of target DNA. Future reductions of the sample volume and optimization of the reaction conditions offer the potential of being able to perform such analyses on as little as a single copy of genomic DNA target. Genomewide association studies are expected to reveal how allelic variation underlies susceptibility to common diseases.1,2 With >10 million common single-nucleotide polymorphisms (SNPs) to consider,3 any large-scale analysis of patient and control populations presents a daunting experimental challenge. Assay cost, complexity, accuracy, and sensitivity are important considerations.4 We have previously described an approach for analyzing SNPs that combines invasive cleavage technology, a very specific nucleic acid assay system, with DNA array technology.5,6 These two technologies, employed in concert, offer a route to the accurate scoring of hundreds of thousands of SNPs. The assay * To whom correspondence should be addressed. Phone: (608) 262-9207. Fax: (608) 265-6780. E-mail: [email protected]. Internet: http://www.chem.wisc.edu/˜smith. (1) Wang, W.; Barratt, B. J.; Clayton, D. G.; Todd, J. A. Nat. Rev. Genet. 2005, 6, 109-118. (2) Kruglyak, L. Nat. Genet. 1997, 17, 21-24. (3) Gunderson, K. L.; Steemers, F. J.; Lee, G.; Mendoza, L. G.; Chee, M. S. Nat. Genet. 2005, 37, 549-554. (4) Melton, L. Nature 2003, 422, 917-923. (5) Lu, M.; Hall, J. G.; Shortreed, M. R.; Wang, L.; Berggren, W. T.; Stevens, P. W.; Kelso, D, M.; Lyamichev, V.; Neri, B.; Skinner, J. L.; Smith, L. M. J. Am. Chem. Soc. 2002, 124, 7924-7931. (6) Olivier, M.; Chuang, L.; Chang, M.; Chen, Y.; Pei, D.; Ranade, K.; Witte, A. D.; Nguyet, J. A.; Pratt, R.; Neri, B.; Wang, L.; David, R.; Cox, D. R. Nucleic Acids Res. 2002, 30, e53.

6594 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

complexity is built into the chip so that the user is required only to add genomic target, enzyme, and reaction buffer. A universal postreaction signal amplification process provides the sensitivity to score SNPs in genomic DNA with no requirement for PCR.7 A key issue in high-throughput SNP analysis is the detection sensitivity. This is important because the amount of human genomic DNA sample available for genetic studies can be very limited. One approach to this problem is to employ preamplification of the DNA sample.8-11 Although such preamplification technologies can be valuable for particular applications, possible biases introduced during the amplification procedure are a concern for many studies. Being able to directly analyze an unamplified genomic DNA sample would eliminate the possibility of a bias or error introduced during the target amplification process. Other issues associated with existing approaches to largescale SNP analysis were discussed in depth in a recent review.12 In our previous work, 1 amol (∼500 ng) of target DNA was employed for genetic analyses using surface-invasive cleavage arrays, an amount of target DNA that is fairly readily obtained from a routine 10-mL blood sample. Although adequate for applications such as clinical diagnostic testing, this is more DNA sample than would be available for many large-scale genetic studies. Pushing the target DNA requirement down to the zeptomole (10-21 mol or 600 molecules) or lower range would comprise a tremendous advance in the power and utility of the technology. In the present work, we demonstrate the ability to detect surface-invasive cleavage reactions by counting individual molecular cleavage events produced on the surface. Rolling circle amplification (RCA) to produce a long single-stranded DNA product,13 used in conjunction with fluorescence staining with the dye SYBR Green I, yields a large fluorescence signal from individual 5′-phosphate groups produced on the surface during (7) Chen, Y.; Shortreed, M. R.; Peelen, D.; Lu, M.; Smith, L. M. J. Am. Chem. Soc. 2004, 126, 3016-3017. (8) Paez, J. G.; Lin, M.; Beroukhim, R.; Lee, J. C.; Zhao, X.; Richter, D. J.; Gabriel, S.; Herman, P.; Sasaki, H.; Altshuler, D.; Li, C.; Meyerson, M.; Sellers, W. R. Nucleic Acids Res. 2004, 32, e71. (9) Tzvetkov, M. V.; Becker, C.; Kulle, B.; Nurnberg, P.; Brockmoller, J.; Wojnowski, L. Electrophoresis 2005, 26, 710-715. (10) Hosono, S.; Faruqi, A. F.; Dean, F. B.; Du, Y.; Sun, Z.; Wu, X.; Du, J.; Kingsmore, S. F.; Egholm, M.; Lasken, R. S. Genome Res. 2003, 13, 954964. (11) Dean, F. B.; Hosono, S.; Fang, L.; Wu, X.; Faruqi, A. F.; Ward, P. B.; Sun, Z.; Zong, Q.; Du, Y.; Du, J.; Driscoll, M.; Song, W.; Kingsmore, S. F.; Egholm, M.; Lasken, R. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5261-5266. (12) Syva¨nen, A. C. Nat. Genet. 2005, 37, S5-10. 10.1021/ac051025p CCC: $30.25

© 2005 American Chemical Society Published on Web 09/15/2005

Figure 1. Schematic diagram of the invasive cleavage assay. The nucleotide at the 3′ end of the invader oligonucleotide is designed to overlap at least one base into the downstream duplex formed by the probe and the target strand. The unpaired region on the 5′ end of the probe, or “flap”, along with an immediately downstream paired nucleotide can then be removed by a class of structure-specific 5′exonucleases. Absolute complementarity between the probe and the target sequence at the position of overlap is required for efficient enzymatic cleavage. In the example above, the wild-type (WT) target has a complementary base to the probe at the point of overlap, which is highlighted in the yellow box. The probe flap is recognized by the FEN enzyme and flap cleavage results. In contrast, the mutant-type (MT) target does not have a complementary base at the point of overlap, and thus, no cleavage occurs.

the invasive cleavage reaction. The number of fluorescent features produced on the surface is dependent upon the concentration of target DNA. Analysis of the results indicates that, under the conditions employed, successive binding and dissociation steps of target DNA molecules to the surface-bound DNA probes effectively “cycles” the target DNA on the surface such that multiple cleavage events are produced per target strand. The combination of target cycling and postreaction signal amplification provides the necessary sensitivity to score SNPs in unamplified genomic DNA by counting the individual molecular cleavage events. EXPERIMENTAL SECTION Invasive Cleavage Reaction. A three-dimensional structure is formed upon hybridization of two partially overlapping synthetic DNA oligomers, known as the upstream oligonucleotide (also referred to as the invader oligonucleotide) and the downstream oligonucleotide (also referred to as the probe oligonucleotide), to a single-stranded target DNA (Figure 1). When the probe and the target have complementary bases at the point of overlap, denoted by the yellow box in Figure 1, then a thermostable flap endonuclease (FEN) enzyme specifically recognizes the threedimensional structure formed and cleaves the unpaired flap from the probe one base downstream from the point of overlap. This produces a free 5′-phosphate, which can be further utilized to generate a detectable signal (see below). When the probe and target do not have complementary bases at the point of overlap, no flap cleavage occurs. The cleavage specificity is ∼300:1 for paired versus unpaired bases.14 This provides the basis for a highly (13) Nallur, G.; Luo, C.; Fang, L.; Cooley, S.; Dave, V.; Lambert, J.; Kukanskis, K.; Kinsmore, K.; Lasken, R.; Schweitzer, B. Nucleic Acids Res. 2001, 29, e118.

specific assay for the detection of DNA sequences.15 In the analytical assay, the reaction temperature is adjusted to match the melting temperature of the probe-target duplex and to be slightly below the melting temperature of the invader-target duplex. Probe oligonucleotides cycle on and off the target, becoming cleaved by the FEN enzyme during the hybridization event. In solution, as many as 3000 probes may be cleaved per target in 90 min.16 Surface-Invasive Cleavage Reaction. The invasive cleavage reaction has been successfully multiplexed using an addressed array format where either the probe or the probe and invader oligonucleotides are covalently attached to a surface. The product of the cleavage reaction, an immobilized probe with a free 5′-phosphate group, is detected using a series of biochemical reactions that includes ligation, rolling circle amplification, and fluorescent dye labeling (Figure 2). First, a primer oligonucleotide is ligated to the immobilized probe using a degenerate guide DNA. The primer is unique in that it yields a free 3′-hydroxyl group, which can be extended by polymerase. A circular template is used in the polymerase reaction, which produces a single-stranded product DNA that is covalently attached at one end to the surface and is several thousand nucleotides in length. The long RCA product is finally labeled with a fluorescent dye, and the chip is imaged using a microscope or fluorescence scanner. Chemical Reagents and Oligomer Sequence. The following sequences were designed for genotyping a single polymorphism (W1282X) in the cystic fibrosis transmembrane conductance regulator gene.17 We note that W1282X is formally a mutation rather than a SNP, as SNPs are defined as polymorphisms for which the minor allele frequency is at least 1%.18 However, as a single-nucleotide variation of substantial medical consequence, it was chosen here as a model system: invader oligonucleotide, 5′GCT CAC CTG TGG TAT CAC TCC AAA GGC TTT CCT A-3′; wild-type probe, 5′-dabcyl-CCA CTG TTG CAA AGT TAT T-(S18)10SH-3′; mutant-type probe, 5′-dabcyl-TCA CTG TTG CAA AGT TAT TG-(S18)10-SH-3′. Synthetic target DNAs were used in these experiments: wild-type target, 5′-GGA TTC AAT AAC TTT GCA ACA GTG GAG GAA AGC CTT TGG AGT GAT ACC ACA GGT GAG CAA AAG-3′; mutant-type target, 5′-GGA TTC AAT AAC TTT GCA ACA GTG AAG GAA AGC CTT TGG AGT GAT ACC ACA GGT GAG CAA AAG-3′; degenerate guide DNA, 5′-NNN NNN GCA TTC CG-3′ (where N ) G, A, T, C). For the initial test of rolling circle amplification on surfaces, a thiol-modified primer with a free 3′-hydroxyl group was used: 5′-HS-TTT TTT TTT TTT TTT TAT GAT CAC AGC TGA GGA TAG GAC ATG CGA-OH-3′. This same sequence was used in the solution-phase RCA reactions. All of the aforementioned sequences were synthesized by the University of Wisconsin Biotechnology center and purified ac(14) Lyamichev, V. I.; Mast, A. L.; Hall, J. G.; Prudent, J. R.; Kaiser, M. W.; Takova, T.; Kwiatkowski, R. W.; Sander, T. J.; de Arruda, M.; Arco, D. A. Nat. Biotechnol. 1999, 17, 292-296. (15) Lyamichev, V. I.; Kaiser, M. W.; Lyamicheva, N. E.; Vologodskii, A. V.; Hall, J. G.; Ma, W. P.; Allawi, H. T.; Neri, B. P. Biochemistry 2000, 39, 95239532. (16) Hall, J. G.; Eis, P. S.; Law, S. M.; Reynaldo, L. P.; Prudent, J. R.; Marshall, D. J.; Allawi, H. T.; Mast, A. L.; Dahlberg, J. E.; Kwiatkowski, R. W.; de Arruda, M.; Neri, B. P.; Lyamichev, V. I. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8272-8277. (17) Chen, Y.; Shortreed, M. R.; Olivier, M.; Smith, L. M. Anal. Chem. 2005, 77, 2400-2405. (18) Brookes, A. J. Gene 1999, 234, 177-186.

Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

6595

Figure 2. Schematic diagram of RCA following surface invasive cleavage. The product of the surface invasive cleavage reaction is a singlestranded oligonucleotide that is covalently attached to a surface at its 3′ end and has a free phosphate group at its 5′ end. A guide DNA with six degenerate bases at its 3′ end is used in the ligation reaction to provide a universal mechanism for detecting all possible cleavage products. The short strand of DNA that gets ligated to the surface-bound DNA is synthesized with two 3′ ends, which are connected by a short spacer. The free end of the ligated sequence contains a primer for rolling circle amplification. Using the phage φ29 DNA polymerase in conjunction with a circular DNA template produces a long single-stranded product DNA, which is covalently attached to the surface. The elongated DNA is noncovalently labeled with a fluorescent dye and detected by fluorescence microscopy. Uncleaved probes do not participate in any of the labeling reactions because their free ends are blocked with a dabcyl moiety.

cording to a procedure that was outlined previously.19 A specially modified 3′-3′ RCA primer (purchased from and PAGE purified by the Yale University Critical Technologies facility), 3′-AGC GTA CAG GAT AGG AGT CGA CAC TAG TAT TTT-(C18)-TTT TCG GAA TGC -3′, was obtained by reversal of backbone polarity during chemical synthesis,20 immediately after the C18 spacer, and was designed to have a priming sequence of 29 bases that is complementary to the circular DNA. In the primer just shown, C18 refers to spacer phosphoramidite 18 (Glen Research Corp., Sterling, VA), which is a hexa(ethylene glycol) linker. The linear precursor for the circular DNA was 5′-PO4-CGC ATG TCC TAT CCT CAG CTG TGA TCA TCA GAA CTC ACC TGT TAG ACG CCA CCA GCT CCA ACT GTG AAG ATC GCT TAT-3′, and the guide DNA for creating the circular RCA template was 5′-AGG ACA TGC GAT AAG CGA TC-3′. The two sequences used for the determination of detection efficiency (mixed 5′-phosphate and 5′-dabcyl) were as follows: 5′-phosphate-TCA CTG TTG CAA AGT TAT TG-(S18)10-SH-3′; 5′-dabcyl-TCA CTG TTG CAA AGT TAT TG-(S18)10-SH-3′. These two oligonucleotides were obtained in PAGE purified form from Integrated DNA Technologies (Coralville, IA). The heterobifunctional linker sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SSMCC) was obtained from Pierce (Rockford, IL). φ29 DNA polymerase and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). (3-Aminopropyl)trimethoxysilane was from Aldrich (Mil(19) Glen Research Corp. User Guide to DNA Modification and Labeling; 1996. (20) Lizardi, P. M.; Huang, X.; Zhu, Z.; Bray-Ward, P.; Thomas, D. C.; Ward, D. C. Nat. Genet. 1998, 19, 225-232.

6596

Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

waukee, WI). SYBR Green I21 was from Molecular Probes (Eugene, OR). SSC buffer was purchased from Ambion (Austin, TX). Preparation of DNA-Modified Glass Substrates. Cover glass slides (25 × 25 mm, Fisher Scientific, Pittsburgh, PA) were rinsed using a standard protocol. The glass slides were immersed in acid solution (H2SO4/H2O2 7:3) at 85 °C for 10-12 h, then rinsed with ultrapure water (>18 MΩ), and dried under a stream of N2 gas. Following incubation in 1% (v/v) (3-aminopropyl)trimethoxysilane (Aldrich) in 95% acetone/water for ∼10 min, the slides were washed several times with acetone and water, dried with a stream of nitrogen gas, and annealed in an 110 °C oven for 45 min.22 The resulting amine-terminated chips were reacted with a 0.4 mg/ mL solution of the heterobifunctional cross-linker SSMCC in 0.1 M pH 7.0 Tris buffer for 15-20 min at room temperature. After rinsing the chips with water, 1 µL of 0.5 mM probe DNA was deposited on the glass chip and allowed to react overnight in a humid chamber.23 The chip was then rinsed with water and dried under a stream of nitrogen. Preparation of DNA-Modified Glass Substrates for Determination of Detection Efficiency. Surfaces for the determination of detection efficiency were prepared as described above except that two probes were mixed together to a final concentration of 0.23 mM prior to deposition on the chip surface. The two (21) Zipper, H.; Brunner, H.; Bernhagen, J.; Vitzthum, F. Nucleic Acids Res. 2004, 32, e103. (22) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456-5465. (23) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044-8051.

probes were terminated either in 5′-phosphate or 5′-dabcyl (see Reagents). The six different ratios (phosphate/dabcyl) employed were 0:1, 1:1, 1:10, 1:100, 1:1000, and 1:10000. Each of the six probe mixtures were spotted onto four separate chips. Determination of Oligonucleotide Hybridization Density on the Surface. A surface modified with single-stranded oligonucleotides was incubated with 2 µM fluorescein-labeled complementary DNA in 2× SSPE/0.2% SDS hybridization buffer for 30 min. The surface was rinsed by placing the chip in the bottom of a 50-mL Falcon tube that was filled with hybridization buffer and agitating it by hand for 5 min. This rinsing process was performed twice on each chip. The chip was then placed in 5 mL of 132 mM KOH/50 mM KCl solution for 5 min to elute the hybridized DNA from the substrate. The concentration of fluorescent oligonucleotides that were eluted from the chip into the 5-mL volume was determined by comparison of its fluorescence signal to that of a series of solutions with known concentrations of fluorescent oligonucleotides. The fluorescence signals from the unknown sample and the calibration solutions were measured simultaneously in a plate reader (Biotek, Flx800, 50 µL/well). The hybridization density was calculated by determining the number of eluted oligonucleotides and dividing that number by the surface area of the chip. Surface Invader Reaction. In surface-invasive cleavage reactions,7 1 µL of synthetic target DNA was added to 10 µL of reaction buffer (250 nM invader oligonucleotide, 10 mM MOPS, pH 7.5, and 7.5 mM MgCl2). The concentration of the added target depended on the experiment (see below). Then, 50 ng of Afu FEN (Cleavase X, Third Wave Technologies, Madison, WI) was added. The glass slides were incubated in a humid chamber at 58.5 °C for ∼3.5 h. The chips were rinsed with 0.1% SDS in 1× SSPE buffer, soaked in 8 M urea for 10 min at 37 °C, and then thoroughly rinsed with a large amount of water. Ligation Reaction. Surface-bound probe oligonucleotides are synthesized with a terminal dabcyl group, which prevents nonspecific ligation. Cleavage of the surface-bound probe produces a free 5′-phosphate group that participates in the ligation reaction. The RCA primer (3 µM) is ligated to the free end of the cleaved probes at 0 °C using 0.3 mM degenerate guide DNA and 1 unit/ µL T4 DNA ligase in buffer solution that is supplied by the manufacturer as part of the ligation kit. The ligation reaction is carried out for 12 h. The chip was then rinsed under a stream of 1× SSC buffer with 0.2% SDS detergent followed by water before being dried under a stream of nitrogen. Rolling Circle Amplification on Surface-Bound Primers. Preparation of the circular RCA template was described previously.20 The circular template (0.1 µM in 10 µL of 50 mM TrisHCl, pH 7.5) was first hybridized with the surface-bound primer at 37 °C for 30 min. The RCA reaction was initiated by addition of 0.5 unit/µL phage φ29 DNA polymerase, 0.2 µg/µL BSA, and 3 mM dNTP in 50 µL of the reaction buffer that was supplied by the enzyme manufacturer. The reaction was performed at 30 °C for up to 17 h. The chip was then soaked in 1× SSC buffer with 0.2% SDS at 60 °C for 30 min. The chip was transferred to a 50mL Falcon tube that was filled with 1× SSC and 0.1% Triton-100. Finally, the chip was rinsed with water. The chips were then incubated with 1× SYBR Green I dye in 1× TBE buffer for 30 min at room temperature.

Rolling Circle Amplification in Solution. The 50-µL solutionphase RCA reaction consisted of the following: 2 pmol of RCA primer; 2 pmol of circular DNA template; 0.5 unit/µL phage φ29 DNA polymerase; 0.2 µg/µL BSA; 3 mM dNTP; 1× φ29 DNA polymerase reaction buffer. The reaction temperature was held constant at 30 °C for 12 h. Fluorescence Microscope. A Pentamax EEV512X512FT intensified CCD camera was mounted to a Nikon Eclipse TE20000U inverted microscope. The camera software controller gain was set at 17, and the hardware intensifier gain was set at 15. The excitation source was a Cyonics argon ion laser operated at 488 nm. Light from the laser was filtered with a 488-nm interference filter (New Focus). The light was then focused with a 30-cm focal length plano-convex lens such that the focal point was at the surface of the glass slides. The microscope objective used was an Olympus PlanApo (oil 1.45 NA, 60×). The objective was optically coupled to the coverslip with immersion oil (type DF, Cargille, Cedar Grove, NJ). Two 488-nm holographic notch filters (Kaiser Optical, HNFP) were used between the objective and the ICCD. Two 525-40 nm band-pass optical filters were employed as emission filters and were installed in the dichroic filter cube. RESULTS AND DISCUSSION Detection of Single RCA Product Molecules in Homogeneous Solution. The detection of RCA products in solution is readily achieved by staining the DNA with fluorescent dye and acquiring images of it with a fluorescence microscope (Figure 3). The DNA polymerase φ29 was used in the amplification reactions that are described in this work. This enzyme is highly processive with excellent strand displacement properties,24 and it adds nucleotides to a primed strand of DNA at a rate of 2280 nt/min at 30 °C.25 It is therefore capable of producing singlestranded DNA products with lengths on the order of 109 or more nucleotides.20 Three dye-labeling schemes were evaluated during the process of optimizing detection sensitivity: hybridizing the single-stranded RCA product with fluorescently tagged complementary DNA;26 employing fluorescently tagged dUTP as a nucleotide in the amplification reaction;27 and staining the RCA product with SYBR Green I. The latter method produced the best contrast under the conditions used here. Although SYBR Green I is generally considered a stain for double-stranded DNA, it does have a reasonable affinity for long single-stranded DNAs. According to a previous report, the binding of SYBR Green I to ssDNA is ∼11fold lower than for dsDNA.21 The labeling ratio of SYBR Green I with dsDNA is 1:6.7 dye molecules:base pairs. Therefore, we estimate a labeling ratio of SYBR Green I with ssDNA of 1:80 dye molecules:bases. Furthermore, background produced by free SYBR Green I dye or by SYBR Green I dye-stained RCA primer and template is small relative to fluorescence from the stained RCA product (Figure 4). An RCA reaction mixture was prepared, which contained 0.5× SYBR Green I dye. The fluorescence of (24) Bernad, A.; Lazaro, J. M.; Martin, G.; Garmenida, C.; Salas, M. J. Biol. Chem. 1989, 264, 8935-8940. (25) Soengas, M. S.; Gutierrez, M. S.; Salas, M. J. Mol. Biol. 1995, 253, 517529. (26) Gerhard, A. B.; Schmidt, T.; Nilsson, M. Anal. Chem. 2004, 76, 495-498. (27) Christian, A. T.; Melissa, S.; Pattee, M. S.; Attix, C. M.; Reed, B. E.; Sorensen, K. J.; Tucker, J. D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14238-14243.

Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

6597

Figure 3. Fluorescence photomicrographs of RCA product molecules that were synthesized in solution. A 100-µL drop of 100 pM RCA product solution was mixed with 1× SYBR Green I (in TBE buffer) and deposited on a clean glass slide. The image was taken with 50-ms exposure time. (A) The molecules are observed freely diffusing in solution. (B) Some of the molecules nonspecifically adsorbed to the glass slide.

Figure 4. Fluorescence photomicrograph of RCA product molecules that were directly synthesized on a glass slide.

Figure 5. Fluorescence photomicrograph displaying the stretched RCA product molecules.

this solution was measured in a fluorescence spectrophotometer prior and after amplification. The fluorescence signal following the reaction was 740-fold brighter than the starting mixture and 2500-fold brighter than a solution of 0.5× SYBR Green I dye in TBE buffer after subtracting the TBE background. RCA Amplification with Immobilized Primers. As described in the Experimental Section, 5′-thiol-modified oligonucleotides were coupled to a glass substrate and the free 3′-hydroxyl was extended using RCA. The fluorescence image obtained from such surface-bound RCA reaction products (Figure 4) is similar in appearance to the image obtained of nonspecifically adsorbed solution-phase RCA products (Figure 3B). However, in this experiment, no freely diffusing RCA products were observed. This indicated that all of the product molecules were attached to the surface. In addition, the primers were localized in ∼3-mm spots rather than distributed across the entire surface and the RCA products were only observed within the spots. There was no nonspecific RCA amplification outside of regions where primer was immobilized. Stretching RCA Product Molecules on a Surface. The stretching of double-stranded DNA on surfaces is well-established and is the foundation of a technique known as optical mapping.28,29

A common method of stretching DNA is to draw a solution across a surface that is capable of adsorbing DNA. The DNA will adsorb at one end and then be stretched in the direction of fluid motion. As it stretches, it adsorbs to the surface and appears as a streak in fluorescence images. A similar experiment was performed here with single-stranded surface-bound RCA products.30,31 After the surface RCA reaction, a 100-µL drop of 1× SYBR Green I with 0.2% SDS was placed on the center of the chip. A coverslip was placed on top. The liquid spreads outwardly from the center of the coverslip to the edge. This fluid motion stretched the surfacebound RCA product in an outward direction and yielded characteristic streaks in the fluorescence image as shown in Figure 5. The longest observed ssDNA molecule was ∼8 µm.32 AFM imaging of the same surfaces also showed stretched DNA molecules of similar length (data not shown). This observation provides strong evidence that the fluorescence features observed on the surface do in fact correspond to RCA-generated DNA strands. Combined Efficiency of Ligation and Rolling Circle Amplification on Immobilized Primers with Free 5′-Phosphate Groups. Ideally, the efficiency of the process employed for

(28) Samad, A. H.; Cai, W.; Hu, X.; Irvin, B.; Jing, J.; Reed, J.; Meng, X.; Huang, J.; Huff, E.; Porter, B.; Shenkar, A.; Anantharaman, T.; Mishra, B.; Clarke, V.; Dimalanta, E.; Edington, J.; Hiort, C.; Rabbah, R.; Skiada, J.; Schwartz, D. C. Nature 1995, 378, 516-517.

6598 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

(29) Guo, X.; Huff, E. J.; Schwartz, D. C. Nature 1992, 359, 783-784. (30) Xavier, M. Nano Lett. 2001, 1, 341-343. (31) Woolley, A. T.; Kelly, R. T. Nano Lett. 2001, 1, 345-348. (32) Mizuta, R.; Mizuta, M.; Kitamura, D. Arch. Histol. Cytol. 2003, 66, 175181.

Figure 6. Images of surfaces with various ratios of 5′-phosphate/5′-dabcyl, which simulated different levels of cleavage in a surface invader reaction. The ratio of phosphate/dabcyl for (A-F) is 0:1, 1:10000, 1:1000, 1:100, 1:10, and 1:1, respectively. Table 1. Numbers of Molecules Detected on Surfaces with Various Ratios of 5′-Phosphate/5′-Dabcyla

a

probe ratio 5′-phosphate/ 5′-dabcyl

5′-phosphate density (probes/cm2)

detected molecules (spots/cm2)

detection efficiency (%) (detected spots/ phosphate)

adjusted detection efficiency (%)

1:1 1:10 1:100 1:1000 1:10000

(1.1 ( 0.2) × 1011 (2.2 ( 0.2) × 1010 (2.2 ( 0.2) × 109 (2.2 ( 0.2) × 108 (2.2 ( 0.2) × 107

saturated saturated (2.3 ( 0.4) × 107 (6.4 ( 0.5) × 106 (1.2 ( 0.2) × 106

1.2 ( 0.2 2.9 ( 0.4 5.5 ( 1.0

1.9 ( 0.2 4.7 ( 0.4 8.8 ( 1.0

Values for detected molecules are the averages and standard deviations from four separate chips.

Table 2. Concentration Dependence of Surface-Invasive Cleavage Reaction with Single-Molecule Detection target amount (amol)

detected cleavage events (2500 µm2 area)

cleavage events/target assume 4.2% det eff

0 1 10 100

4.0 ( 1.0 8.7 ( 1.5 30.7 ( 3.2 242.3 ( 12.0

7.9 ( 1.7 4.3 ( 0.8 3.8 ( 0.6

labeling 5′-phosphate groups produced on the surface by the invasive cleavage reaction would be 100%. However, the approach described here consists of successive steps of ligation, RCA, fluorescence labeling, and microscopic imaging, each of which might well be expected to have efficiency less than unity. We wished to evaluate the overall detection efficiency of the process and, to the extent possible, understand how this overall efficiency related to the efficiencies of each step in the process. This was studied using probe-modified surfaces to simulate the results of the surface invader reaction. Surfaces were prepared using mixtures of oligonucleotides that were terminated at their 5′ ends

either with dabcyl or with phosphate. The phosphate-terminated probes are identical to the products of cleavage in the surface invader reaction. The dabcyl-terminated probes are identical to the noncleaved probes used as starting material. The fraction of phosphate-terminated probe relative to dabcyl-terminated probe provides a simulated level of cleavage. All surfaces were prepared using the same total probe concentration. The number of hybridizable probes on the surfaces was determined using fluorescence wash-off experiments (Experimental Section and ref 33) and was found to be 2.2 × 1011molecules/cm2. Five surfaces were analyzed with ratios of phosphate/dabcyl of 1:1, 1:10, 1:100, 1:1000, and 1:10000 (Figure 6). Randomly chosen regions from each of the images displayed in Figure 6 were selected for analysis. Bright spots, assumed to be the products of the ligation and RCA reactions, were counted (Table 1). Objects taken as single molecules in the various acquired images had S/N ratios in the range of 50-100. For the purpose of counting, image contrast was adjusted so that bright objects appeared white and weakly fluorescent objects appeared black. Bright spots were then visually counted. To guard against (33) Peelen, D.; Smith, L. M. Langmuir 2005, 21, 266-271.

Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

6599

Figure 7. Fluorescence photomicrographs from surface-invasive cleavage reactions using mutant-type synthetic DNA target followed by rolling circle amplification. (A) Mutant-type probe chip; (B) wild-type probe chip.

photobleaching, chips were not exposed to light prior to imaging. Sample chips were brought into focus on the microscope using light that was only incident on a very small region of the surface. Light was turned off and the chip was moved laterally so that an unexposed region of the surface would be imaged. Excitation light was synchronized with image acquisition. Hence, no photobleaching occurred and detection efficiency was unaffected. Detection efficiency is defined as the percentage of free phosphate groups that produced a detectable fluorescence feature. The unadjusted range of apparent detection efficiencies fell between 1.2 and 5.5% and appeared to increase with decreasing density of phosphate. One factor to consider is that probes on these surfaces are somewhat unstable to the conditions of the surface-invasive cleavage reaction. In particular, the long incubations at elevated temperature (58.5 °C for 3 h) cause a small fraction of the probe molecules to fall off of the surface. This issue is being addressed as part of other studies.34,35 To compensate for this loss of probes in calculating detection efficiency, probe loss was measured on chips prepared in a fashion identical to the process used for determining detection efficiency. One of two chips prepared was subjected to the conditions of the surface invader reaction (3.5-h incubation in MOPS buffer at 58.5 °C), and one chip was stored at room temperature in TEA buffer. Hybridization density on the two chips was analyzed as described above. The hybridization density dropped by 38% following the high-temperature treatment. Detection efficiencies adjusted for this loss in probe density are shown on the far right column of Table 1. Target Concentration Dependence of Detection Following Surface Invader Reaction. The surface invader reaction was performed using four different amounts of synthetic target (0, 1, 10, and 100 amol) (Table 2). The data in Table 1 suggest that detection efficiency is a function of surface 5′-phosphate density. Detection efficiency increases as the fraction of free phosphate groups decreases. This fact complicates the analysis of cleavage events per target (target cycling) because knowledge of the detection efficiency is necessary for the calculation. A precise calculation of target cycling would require a variable detection efficiency, whose value is in turn dependent upon the level of (34) Lu, M.; Knickerbocker, T.; Cai, W.; Yang, W.; Hamers, R. J.; Smith, L. M. Biopolymers 2004, 73, 606-613. (35) Yang, W.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253-257.

6600

Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

cleavage (i.e., upon the density of free 5′-phosphate groups). For the time being, we have taken an approximate value of 4.2% for the detection efficiency, which corresponds to a ratio of cleaved to intact probe oligonucleotides of 1:1000 (Table 1 and Figure 6C). Further studies are in progress to refine the analysis. The main conclusion that can be drawn from the data in Table 2 is that there are clearly multiple cleavage events per target. It is also apparent that more work needs to be done to increase target cycling, which will concomitantly improve detection sensitivity. Specificity of the Surface Invader Reaction. Two glass slides were prepared and functionalized with oligonucleotide probe sequences specific for either mutant or wild-type w1282x target. Mutant-type synthetic DNA target (100 amol) was added to both chips, and the invader reaction was carried out and followed by ligation, RCA amplification, and imaging (Figure 7). The ratio of amplification products counted on the two chips was 30:1 (mutant/ wild type). The background intensity in these images was not systematically investigated, but it can be ascribed to any number of possible sources (probe, enzyme, buffer, substrate, etc.). The intensity of the fluorescent RCA-generated features is ∼100 fold greater than the background fluorescence level. CONCLUSION The present work provides a proof-of-principle demonstration of the direct analysis of single-nucleotide polymorphisms with detection of individual DNA cleavage events on surfaces. This work is directed toward development of the ability to detect the cleavage events produced by a single molecule of target nucleic acid. ACKNOWLEDGMENT We thank Third Wave Technologies (TWT) for providing the FEN enzyme and the design of the invasive cleavage assay for the CF mutations W1282X. We also thank Professor James Weisshaar for the use of the Nikon microscope and Dr. Yan Chen for many helpful discussions. L.M.S. has a financial interest in TWT. This work was supported by NIH grants R01HG02298 and R01HG003275. Received for review June 9, 2005. Accepted August 11, 2005. AC051025P