Patent Application
20170275680
The present invention relates to methods and apparatus for nucleic acid amplification, hybridization and microarray analysis.
Nucleic acid diagnostics is currently the fastest growing segment of the in vitro diagnostics market. However, with the perspective of personalized medicine in the future, these diagnostic techniques must be simple, fast, and especially, reliable. DNA hybridization is a promising tool for nucleic acid diagnostics because the method is simple and has a high sample-throughput potential. However, hybridization assays are limited by an inherently low specificity, which is the main cause of discrepancies in the assay results. This limitation is aggravated for single nucleotide polymorphism (SNP) analysis in which the mismatched target strand (MM) varies from the perfectly matched target (PM) strand by only a single base pair. In order to improve the assay specificity, DNA hybridization, or subsequent wash step, is conventionally conducted at stringent conditions, when high temperature, low ionic strength or chemically denaturing medium is applied to reduce the nonspecific signal. These stringent conditions bring the duplexes near their melting temperatures, where a marginal difference in the duplex stability (i.e. PM vs. MM) causes a significant variation in their affinities. However, this high-temperature method is not effective when conducted for highly multiplexed analyses, such as DNA microarrays where many thousands of targets, each with its own melting temperature, have to be analysed simultaneously at a single optimized temperature. Therefore, low specificity with false-positive and false-negative outcomes is resulted for those targets with melting temperatures far from the hybridization temperature. These faults are widely agreed to be the main pitfall to impact the accuracy of the DNA microarray platform, resulting in a barrier for its adoption for clinical applications.
Various novel methods to boost specificity are reported in which special hybridization probes are designed to work at temperatures well below the melting temperatures. For instance, when oligonucleotide probes with short lengths are used, the nonspecific binding is thermodynamically less favorable, leading to an improved sensitivity while boosting specificity. However, the design of these special probes is usually complicated and the method of using them is not compatible with multiplex analyses.
Additionally, nucleic acid amplification is an integral part of molecular diagnostics. The polymerase chain reaction (PCR), invented in 1980s, made a significant contribution in the area of molecular biology and molecular diagnostics. PCR is a powerful technique and is still considered the gold standard for nucleic acid amplification. However, the need for thermocycling in PCR limits its use in certain settings (e.g. in limited-resources or point-of-care environments). An alternative to thermocycling is the isothermal method, which includes a variety of techniques such as loop-mediated amplification (LAMP), rolling-circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), and helicase-dependent amplification (HDA). HDA, which uses helicase instead of heat to denature double-stranded DNA, is considered a true isothermal technique because the entire process occurs at a single temperature. However, the rate-limiting step of HDA is denaturation, and so the method is limited by the low denaturation efficiency of the helicase. This limitation is supported in the literature, showing that HDA has been successfully used to amplify more for the shorter bacterial DNA and viral cDNA, and less for the longer DNA, such as human DNA.
Based on the foregoing, it would be desirable to provide improved nucleic acid hybridization and microarray methods and apparatus. For example, it would be desirable to provide nucleic acid hybridization and microarray methods and apparatus with enhanced specificity of nucleic acid hybridization without reducing detection sensitivity.
It would also be desirable to provide improved isothermal methods for nucleic acid amplification. For example, it would be desirable to provide HDA (helicase-dependent amplification) methods with improved efficiency and sensitivity.
One aspect of the invention provides a method for nanoparticle-assisted nucleic acid hybridization analysis. An example embodiment of the nucleic acid hybridization method comprises a number of steps. In a first step, probe nucleic acid molecules are immobilized on a surface. The surface may be a solid surface, or a semi-solid surface. For example, the surface may be a gel, polyacrylamide, agar, agarose, or gelatin. The surface may be made of solid or curable materials, for example, glass, silicon, plastic, polymer, cellulose, etc. The solid surface may be, for example, a solid surface inside a test tube or a microfluidic channel, or on a glass slide, a test chip, a microarray chip, a microtiter plate, a nylon membrane, or a film. The surface may be substantially flat, or curved, for example, the surface of a well of a microtiter plate, or the surface (hereafter refer as crust) of a spherical bead. The surface may also be coated or conjugated with one or more compounds, for example, the surface may be aldehyde-functionalized.
There are a number of ways to immobilize or tether probe nucleic acid molecules on the surface. One approach is in situ synthesis, wherein probe nucleic acid molecules are synthesized directly base by base on the surface. Another approach is to spot or print the probe nucleic acid molecules on the surface using contact or non-contact printing methods. Other methods of immobilizing probe nucleic acid molecules are known to persons skilled in the art. For example, immobilization can be achieved by chemical, mechanical, or biochemical methods such as covalent binding, adsorption, polymer encapsulation and so forth. One example method of chemical immobilization is Schiff-base linkage formed between an aminated DNA or oligonucleotides probe and an aldehyde-functionalized glass surface.
The probe nucleic acid molecules are typically single-stranded, or comprise at least a single-stranded region. In some embodiments, the probe nucleic acid molecules may also comprise a double-stranded region, or a triple-stranded region. The probe nucleic acid molecules may be formed from oligonucleotides, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide nucleic acid (PNA). They may include both natural or artificial or synthetic nucleic acids. They may include genomic DNA or even a chromosome preparation (e.g., a chromosome preparation suitable for fluorescent in situ hybridization (FISH)). They may be synthesized or generated or amplified using standard procedures known to those skilled in the art or ordered from commercial vendors. Standard molecular biology methods for probe preparation can be found in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, 3rd edition, which is hereby incorporated by reference. In some embodiments, the probe nucleic acid molecules are between 10 and 1000 nucleotides in length. In some embodiments, the probe nucleic acid molecules are between 10 and 100 nucleotides in length. In some embodiments, the probe nucleic acid molecules are between 10 and 50 nucleotides in length.
After probe immobilization, in a second step, target nucleic acid molecules are flowed to the immobilized probe nucleic acid molecules on said surface in a hybridization buffer solution. The target nucleic acid molecules may be provided in the hybridization buffer solution, and the solution may be allowed to incubate on said surface for a period of time. This incubation will allow the hybridization of the target nucleic acid molecules with the immobilized probe nucleic acid molecules.
The target nucleic acid molecules may be oligonucleotides, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or an artificial or synthetic nucleic acid. They may be synthesized or generated or amplified using standard procedures known to those skilled in the art or ordered from commercial vendors. The target nucleic acid molecules may be isolated directly from samples (e.g. cells, tissues, cell extracts, tissue culture media, bodily fluids, environmental samples, other biological samples etc.), or they may first be amplified by polymerase chain reaction (PCR) or reverse-transcription PCR (RT-PCR) or an isothermal amplification method. In some embodiments, the target nucleic acid molecules may be amplicons amplified from a nucleic acid source (e.g., genomic DNA) using polymerase chain reaction (PCR) or an isothermal amplification method. An example isothermal amplification method may be a nanoparticle-assisted isothermal amplification method, which will be described in this disclosure. The target nucleic acid molecules may comprise synthetic, natural, or structurally modified nucleoside bases. The target nucleic acid molecule can also be from any source organism (e.g., human or another animal, virus, bacteria, insect, plant, etc.).
In some embodiments, the target nucleic acid molecules are between 10 to 1000 nucleotides or base-pairs in length. In some embodiments, the target nucleic acid molecules may exceed 1000 nucleotides or base-pair in length. The target nucleic acid molecules may comprise single-stranded molecules, or double stranded molecules, or combinations thereof. In some embodiments, the target nucleic acid molecules may comprise a single-stranded region and a double-stranded region. The target nucleic acid molecules may be purified or isolated molecules, or may be present in a solution or sample that comprises other molecules or contaminants. The target nucleic acid molecules may comprise nucleic acid molecules having different sequences (e.g., a mixture of genomic DNA molecules, a mixture of different PCR products, a mixture of cDNA molecules, or a mixture comprising two related DNA sequences differing by a single base-pair). The target nucleic acid molecules may be from a single sample source or may be from two or more sample sources (e.g., pooled cDNA molecules from two types of cells, one being stem cell, the other being differentiated cell, or genomic DNA from two human individuals).
The target nucleic acid molecules can either be unlabeled or they can be conjugated or otherwise coupled to a detectable label. Suitable detectable labels include, without limitation, fluorescent labels, redox (electrochemical) labels, and radioactive labels.
Coupling of a fluorescent label to nucleic acid molecules can be achieved using known nucleic acid-binding chemistry or by physical means, such as through ionic, covalent or other forces well-known in the art (see, e.g., Dattagupta et al., Anal. Biochem. 177:85-89 (1989): Saiki et al. Proc. Natl. Acad. Sci. USA 86:6230-6234 (1989); Gravitt et al. J. Clin. Micro. 36:3020-3027 (1998), each of which is hereby incorporated by reference in its entirety). Either a terminal base or another base near the terminal base can be bound to the fluorescent label. For example, a terminal nucleotide base of the target nucleic acid molecules can be modified to contain a reactive group, such as (without limitation) carboxyl, amino, hydroxyl, thiol, or the like.
The fluorescent label can be any fluorophore that can be conjugated to a nucleic acid and preferably has a photoluminescent property that can be detected and easily identified with appropriate detection equipment. Exemplary fluorescent labels include, without limitation, fluorescent dyes, semiconductor quantum dots, lanthanide element-containing complexes, and fluorescent proteins. Example fluorescent dyes include, without limitation, Calcein, FITC, Alexa™, Rhodamine 110, 5-FAM, Oregon Green™ 500, Oregon Green™ 488, RiboGreen™, Rhodamine Green™ Rhodamine 123, Magnesium Green™, Calcium Green™, Cy3™, Alexa™ 546, TRITC, Magnesium Orange™, Phycoerythrin R&B, Rhodamine Phalloidin, Calcium Orange™, Pyronin Y, Rhodamine B, TAMRA, Rhodamine Red™, ROX, Nile Red, YO-PRO™-3. R-phycocyanin, C-Phycocyanin, Cy5™, Thiadicarbocyanine, and Cy5.5™. Other dyes now known or hereafter developed may similarly be used.
Buffer conditions for hybridization are well-known to those skilled in the art and can be varied within relatively wide limits.
After hybridization of target nucleic acid molecules with probe nucleic acid molecules, in a third step, said surface is washed with a wash solution which comprises nanoparticles. The nanoparticles should be in suspended, non-aggregated form, or de-aggregated under suitable conditions. The nanoparticles may be sized between 1 and 100 nanometers. They may be spherical or rod-shaped or of other shapes. The nanoparticles may be coated with negatively charged ions. The negatively charged ions may help prevent aggregation of nanoparticles. The nanoparticles may be formed of a metal, a semiconductor, or an uncharged substrate, such as glass, or combinations thereof. The nanoparticles may be sized between 1 and 50 nm, or between 20 and 30 nm, or between 10 to 20 nm, or between 1 to 10 nm, or between 3.5 to 6.5 nm. The nanoparticles may have a mean particle size of 5.0 nm. The nanoparticles may have a coefficient of variance of particle size that is less than 15% of the mean particle size.
In some embodiments, the crusts of the nanoparticles are loaded with oligonucleotide stabilizers whose sequences are unrelated to the sequences of the probe nucleic acid molecules or the target nucleic acid molecules. The length of the oligonucleotide stabilizers may be 20-mer or shorter, or 15-mer or shorter, or 12-mer.
The concentration of the nanoparticles in the wash solution is in a range of 2 to 20 nM. In some embodiments, the concentration of NaCl in the wash solution may be in a range of 50 to 300 nM. In some embodiments, the wash solution has an ionic strength equivalent to NaCl concentration of between 50 and 150 nM.
In some embodiments, the washing step is performed at an ambient temperature. In some embodiments, the washing step is performed at a temperature below 30° C. In some embodiments, the washing step is performed at a temperature between 20° C. and 25° C.
The metal nanoparticles may be formed of a conductive metal or metal alloy that allows a nanoparticle to be capable of non-covalently associating with a single-stranded nucleic acid molecule. It should be appreciated that the colloidal suspension should maintain the metal nanoparticles in a stable environment in which they are substantially free of aggregation. The metal nanoparticles should not significantly associate with double-stranded nucleic acid molecules. Example metal nanoparticles include, without limitation, gold nanoparticles (AuNPs), silver nanoparticles, platinum nanoparticles, mixed metal nanoparticles (e.g., gold shell surrounding a silver core), and combinations thereof. The metal nanoparticles may be magnetic or magnetically attractable, for example, formed of an inner core such as cobalt and an outer layer such as gold.
Preparation of colloidal metal nanoparticle suspensions can be carried out according to known procedures, e.g., Grabar et al. Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety. Metal nanoparticles may be stabilized in the solution by negatively charged anions, such as citrate, acetate, carbonate, phosphate, oxalate, sulfate, or nitrate.
In some embodiments, the nanoparticles comprise gold nanoparticles. Preparation of gold nanoparticles can be carried out according to known procedures, e.g., J. Turkevich, P. C. Stevenson, J. Hillier, Discuss. Faraday. Soc. 1951, 11, 55-75: J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot. A. Plech. J. Phys. Chem. B 2006, 110, 15700-15707; G. Frens, Colloid & Polymer Science, 1972, 250, 736-741; G. Frens, Nature (London). Phys. Sci. 1973, 241, 20-22; J. W. Slot and H. J. Geuze, Eur. J. Cell Biol. 38, 87 (1985); M. C. Daniels and D. Astruc. Chem. Rev. (Washington D.C.) 104, 293 (2004), each of which is hereby incorporated by reference in its entirety. Briefly, gold nanoparticles are typically produced in a liquid by reduction of chloroauric acid (HAuCl4). After dissolving HAuCl4, the solution is rapidly stirred while a reducing agent is added. This causes Au(III) ions to be reduced to neutral gold atoms. As more and more of these gold atoms form, the solution becomes supersaturated, and gold gradually starts to grow in the form of sub-nanometer particles. The rest of the gold atoms that form stick to the existing particles, and, if the solution is stirred vigorously enough, the particles will be fairly uniform in size. The anions in gold nanoparticle preparation also prevent the gold nanoparticles from aggregating. These anions may include citrate, acetate, carbonate, phosphate, oxalate, sulfate, or nitrate. Alternatively, the nanoparticles can be purchased from commercial sources. For example, gold nanoparticles can be purchased from Sigma Life Sciences.
Although some of the examples in the present disclosure describe experiments performed using gold nanoparticles, it will be appreciated by those skilled in the art that other nanoparticles having similar properties may also be used. For example, silver nanoparticles have been shown to behave like gold nanoparticles in terms of non-covalent binding with single-stranded nucleic acid molecules: Chen et al., Analyst, (2010), 135, 1066-1069, which is hereby incorporated by reference.
After washing, in a fourth step, a determination is made as to whether at least some of the target nucleic acid molecules have hybridized with probe nucleic acid molecules to form a hybridization duplex comprising a strand from the target nucleic acid molecules and a strand from the probe nucleic acid molecules and the level of hybridization. In some embodiments, the surface is dried before a detection method is applied. In some embodiments, a detection method may be applied without drying the surface. This determination or detection may be qualitative or quantitative. A large number of methods are available to detect or quantify hybridization duplexes on the surface. For example, if the target nucleic acid molecules were fluorescently labeled, the surface can be scanned for fluorescence emissions. For example, a confocal laser fluorescent scanner may be used. If both the target and the probe are fluorescently labeled, a detection method called fluorescence resonance energy transfer (FRET) may be used. Alternatively, if the target nucleic acid molecules comprise redox labels, or radioactive labels, other methods may be used to detect the level of hybridization. These methods are well known to those skilled in the art.
One aspect of the invention provides a method for distinguishing two target nucleic acid molecules whose nucleotide sequences differ by at least one nucleotide, the method comprising: carrying out two separate nucleic acid hybridization assays in parallel, the first assay with a first target and a probe, the second assay with a second target and the same probe, each assay comprising: providing a sample solution comprising a target nucleic acid; incubating said sample solution with probe nucleic acid molecules which are immobilized on a surface; washing said surface with a wash solution which comprises nanoparticles; and detecting the presence of target:probe duplex on the surface; whereby the two target nucleic acid molecules are distinguished by different degrees of hybridization to the probe.
The nanoparticle-assisted hybridization method can also be applied to microarray technology. A microarray is a multiplex technology commonly used in molecular biology. Procedures for microarrays are well-known to those skilled in the art: e.g., David Bowtell and Joseph Sambrook, DNA Microarrays: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press; 1st edition (2002), which is hereby incorporated by reference. A microarray consists of an arrayed series of tens, hundreds, thousands, or even tens of thousands of microscopic spots of picomoles (10−2 moles) of oligonucleotides or DNA probes, each having a specific nucleotide sequence. These can be a short section of a gene or other DNA element that are used to hybridize a sample (e.g. cDNA or genomic DNA or RNA). Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the sample. Since an array can contain tens, hundreds, thousands, or even tens of thousands of probes, a microarray experiment can accomplish many tests in parallel.
One aspect of the invention provides a microarray method, the method comprising: providing a solid support; immobilizing a plurality of nucleic acid probes at discrete positions on the support; exposing a sample solution to the probes, the sample solution comprising sample nucleic acid molecules; washing off the sample solution with a wash solution which comprises nanoparticles; and determining the degree of hybridization between the sample molecules and the probes.
One aspect of the invention provides nanoparticle-assisted hybridization methods in association with a microfluidic microarray assembly (MMA) or a microchannel plate assembly. MMA and microchannel plate assemblies are described in WO 2006/060922 and L. Wang and P. C. H. Li, J. Agric. Food. Chem. 55, 10509 (2007), which are hereby incorporated by reference in their entirety. It should be noted that both MMA and microchannel plate assembly can be considered to be a subset of microarrays, and that microchannel plate assembly can be considered to be a subset of MMA.
In an embodiment, a method of using a microfluidic microarray assembly (MMA) comprises: providing a test chip; providing a first channel plate sealingly connectable to said test chip for applying at least one probe reagent to said test chip, wherein said first channel plate comprises a plurality of first microfluidic channels configured in a first predetermined reagent pattern; assembling said first channel plate to said test chip; flowing said at least one probe reagent through said first microfluidic channels to form a first array of said at least one probe reagent on said test chip in said first predetermined reagent pattern; immobilizing said at least one probe reagent on said test chip; removing said first channel plate from said test chip; providing a second channel plate sealingly connectable to said test chip for applying at least one sample reagent to said test chip, wherein said second channel plate comprises a plurality of second microfluidic channels configured in a second predetermined pattern differing from said first predetermined pattern; assembling said second channel plate to said test chip; flowing said at least one sample reagent through said second microfluidic channels to form a second array, wherein said second array intersects said first array at said test locations; flowing a wash solution which comprises nanoparticles through said second microfluidic channels; and detecting any hybridization products at said test locations.
In some embodiments, a plurality of different probes is used, and each of those probes is flowed through separate ones of the first microfluidic channels. In some embodiments, a plurality of different test samples are used, and each of those test samples is flowed through separate ones of the second microfluidic channels. In some embodiments, the first predetermined reagent pattern is a radial pattern and the second predetermined reagent pattern is a spiral pattern. In some embodiments, the first predetermined reagent pattern is a spiral pattern and the second predetermined reagent pattern is a radial pattern.
One aspect of the invention provides nanoparticle-assisted nucleic acid amplification methods. The methods may be isothermal amplification methods, wherein the nucleic acid is amplified at an isothermic temperature that does not require a thermal cycler. The isothermal amplification methods may be nanoparticle-assisted helicase-dependent isothermal nucleic acid amplification methods.
In an example embodiment, the nanoparticle-assisted helicase-dependent nucleic acid amplification method comprises these steps. First, double stranded substrate nucleic acid molecules are denatured by a helicase in a reaction solution which comprises nanoparticles. Then, primers are annealed to the denatured substrate nucleic acid molecules and are extended by a suitable polymerase to produce double-stranded nucleic acid molecules. The newly synthesized double-stranded nucleic acid molecules are then used as substrates by the helicase, entering the next round of the reaction. Thus, a chain reaction develops, resulting in exponential amplification of the substrate nucleic acid molecules. In some embodiments, the substrate nucleic acid molecules are digested with a suitable restriction enzyme to reduce the fragment size of the substrate nucleic acid molecules prior to denaturation by the helicase. For example, the substrate nucleic acid molecules may be digested by a restriction enzyme to a fragment size of less than 500 bp.
In conventional HDA (helicase-dependent amplification), helicase denatures dsDNA before DNA extension, and the rate of this method is limited by helicase's low denaturation efficiency. In this disclosure, we describe nanoparticle-assisted HDA (nanoHDA) which enhances the denaturation efficiency of conventional HDA by using nanoparticles (e.g., AuNPs). Nanoparticles with preferential affinity to ssDNA are utilised to improve helicase-mediated DNA denaturation. The same affinity of nanoparticles can also explain our observation that nanoparticles enhanced specificity by suppressing the formation of primer-dimers.
One aspect of the invention provides a combined method which couples a nanoparticle-assisted helicase-dependent isothermal nucleic acid amplification method with a nanoparticle-assisted nucleic acid hybridization method. The combined method comprises: amplifying a substrate nucleic acid in a helicase-dependent amplification (HDA) reaction in a reaction solution which comprises nanoparticles; purifying the amplified nucleic acid molecules; and using the amplified nucleic acid molecules as target molecules in a nanoparticle-assisted nucleic acid hybridization reaction (which is described in this disclosure). The concentration and other parameters of the nanoparticles used in the nanoHDA reaction and the nanoparticle-assisted nucleic acid hybridization reaction may be different and can be independently optimized.
In drawings which show non-limiting embodiments of the invention:
Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well-known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
A microfluidic bioarray technique has been developed, and this technique uses gold nanoparticles (AuNP targets) for specific detection of single nucleotide polymorphism (SNP). In this technique, no temperature stringency is required, and high specificities in hybridization are achieved by loading the target strands on the crusts of small gold nanoparticles (AuNPs) prior to their hybridization to the oligonucleotide probes immobilized on the microfluidic channel surfaces. Our kinetic studies of DNA hybridization using surface plasmon resonance (SPR) spectroscopy has showed that AuNPs enhanced the dehybridization of the mismatch (MM) duplexes more than that of perfectly matched (PM) duplexes, thus accounting for most of the SNP discrimination power of the AuNP-enabled technique. However, the AuNP targets result in lower hybridization signal intensities than the free target counterparts (See
As inspired from our kinetic analysis, we know that the influence of AuNPs is predominantly on dehybridization. Therefore, we attempt to develop a method to use AuNPs in the washing (dehybridization) step, rather than using them in the hybridization step. In this method, a buffer solution containing AuNPs (5-nm diameter or some other suitable size) is used to flow over the surface-bound duplexes for the removal of the hybridized oligonucleotides by washing (
For nucleic acid analysis, we have previously developed a CD-like chip for microfluidic DNA hybridization that provides the advantage of fast analyses and multiplex capability. This platform, termed as CD-like NanoBioArray chip or CD-NBA chip, utilizes the centrifugal force in order to flow in the target solutions within the microfluidic channels. As shown in
where Spm and Smm are signal intensities at the PM and MM patches, respectively. In DNA microarrays, the nonspecific signals are inevitably detected and they are conventionally reduced by conducting a stringent wash subsequent to DNA hybridization. To compare the methods of AuNP wash and stringent wash, we flow the hybridization buffer (SSC 1×) in the spiral channels at room temperature, with or without AuNPs, respectively. The stringent wash only results in a specificity of 1.3 (compared to 1 in “no wash” channels). On the other hand, the use of AuNPs in the wash step helped to improve specificity to 2.6 but it was only in the presence of AuNPs of 5 nm, but not of 10, 12 and 20 nm, in diameter that the specificity was enhanced by washing (˜2.6). This result is in agreement with the previous observation, in which AuNPs have been used in the hybridization step.
In the stringent wash method, high-temperature or/and low-salt conditions are used to create a destabilizing environment for the formed duplexes and accelerate their dehybridization. This method aims to remove the nonspecific duplexes more than their specific counterparts, thus enhancing the specificity. We compare the AuNP wash and the stringent wash methods directly.
where Spm is the PM signal intensities at different washing condition; is the specificity (calculated by Eq. 1) at each washing condition;
While r=0 shows no correlation or the situation when specificity is achieved without a loss in signal, r=−1 shows the highest anticorrelation between the signal and specificity. From the signals and specificities shown in
The difference between the r values obtained from AuNP wash and stringent wash is also illustrated in our analysis of ˜400 hybridization patches obtained by both methods.
The outcome of high σ and minimal loss in SNR observed with the AuNP wash method can be explained in terms of the dehybridization rate constant kd, which is experimentally determined from our kinetic analyses using SPR spectroscopy. As shown in Table 1, for the MM duplex the kd value (in 10−4 s−1) is observed to enhance by five times, i.e. from 3.2 for stringent wash to 15.9 for AuNP wash at 22° C. On the other hand, the kd value (in 10−4 s−1) for the PM duplex has not increased much, i.e. from 1.7 for stringent wash to 3.0 for AuNP wash. This increase in the kd value for AuNP wash (less than two-fold) is much smaller than the corresponding increase for 40° C. stringent wash (five-fold). This observation is attributed to the enhanced dehybridization of the MM duplexes by AuNPs. On the other hand, increasing the stringent wash temperature from 22° C. to 40° C. enhanced the kd values of both MM duplexes and of PM duplexes, showing the undesirable destabilization of PM duplexes, in addition to the desirable destabilization of MM duplexes. These observations explain our findings obtained in the CD-NBA chip that the PM signals are not affected as much as the MM counterparts in the AuNP wash method, because of the enhanced destabilization of the MM duplex, but not of the PM duplex, leading to the preservation of the signal.
aAll standard errors are determined from two measurements each including five different target concentrations of 10, 20, 40, 80 and 160 nM.
We attribute the difference in enhanced destabilization of the MM duplexes, observed for AuNP wash, compared to stringent wash, to the specific mechanism on which the AuNP wash technique is based. During dehybridization, AuNPs bind to the ssDNA segments (bubbles), which have constantly formed by thermal breathing. The presence of a mismatch base pair, through a cooperative effect, causes weakening and disruption of the neighboring base pairs. In 2006, Zeng and coworkers compared the dissociation curves obtained from PM and MM duplexes, and found that the amount of bubbles was drastically enhanced in the presence of a single MM site in the middle of the duplex. The greater amount of bubbles in the MM duplexes makes them susceptible to the binding by AuNPs, leading to the success of the AuNP wash method. The AuNP wash method target these bubbles in MM duplexes for their enhanced dehybridization or destabilization, to a much larger extent than in the case of PM duplexes. This targeted mechanism of destabilization of MM duplexes causes an enhancement in the specificity without reducing the signal, leading to the observed low negative r value or almost no anticorrelation between signal and specificity. On the other hand, the stringent wash method has similar destabilizing influences on both of the PM and MM duplexes, which lead to their similar extent of accelerated dehybridization and the observed high signal/specificity anticorrelation, or high negative r value.
The preserved sensitivity upon enhancement of specificity is an exclusive feature of the AuNP wash method. This feature was not achieved in the previous AuNP-enabled method, in which AuNP was used in the hybridization step but not in the wash step. In the previous method, the hybridization signals obtained from DNA targets that are conjugated to AuNPs (AuNP targets) were observed to be lower than the signals from free targets, and this observation is attributed to the low hybridization rate constants (kh) of DNA targets, when conjugated to AuNPs. The experiment has been repeated in the CD-NBA chip and shown in
In order to optimize the AuNP wash method, we evaluate the effect of different experimental factors including the salt content of the buffer medium, the length and concentration of the oligonucleotide stabilizer (used to prevent AuNPs in the wash buffer to aggregate) on the performance of the method. Optimization of these factors can improve the effectiveness of AuNP destabilization of MM duplexes, and thus the efficacy of the method.
The histogram in
Using the data in
In order to stabilize AuNPs in the wash buffer against salt-induced aggregation, the AuNP crusts have been loaded with oligonucleotide stabilizers with sequences non-complementary to the probe/target sequences. The aggregation would have happened to the pristine nanoparticles due to high salt contents in the wash buffer. Here, we investigate the effects of length and concentration of the oligonucleotide stabilizers on the specificities obtained in the AuNP wash method.
In order to investigate the applicability of the AuNP wash technique for use with genomic samples, we first evaluated the robustness of the technique upon sequence variation (i.e. the purine content), and then we evaluated the performance of the technique using PCR amplicons as the target strands.
In order to evaluate the robustness of the AuNP wash technique, we employ 3 sequences related to KRAS gene (A20, A60, W20), and two sequences related to a fungal pathogen (B21, NB21); see Table 2. In W20 and A60 targets, the 20 bases of the target that hybridize with the probes are similar to A20 except for variations in the type of the mismatch base-pair (C-C base-pair in A20 and A60 vs. G-G in W20) and also in the length of the target (60 bases in A60 vs. 20 bases in A20). As shown in
We have also used the AuNP wash method to detect single nucleotide polymorphisms (SNPs) in genomic samples, which consists of 4 different alleles of KRAS gene codon 12. The detection of these SNPs is critical for clinicians to choose the appropriate type of therapy for colorectal cancer patients [31].
We have developed a technique for the enhancement of the specificity of DNA hybridization without reducing the signal. This technique is called AuNP wash, which may be performed in a CD-NBA chip using a buffer solution containing 5-nm gold nanoparticles (AuNPs). The solution dynamically washes the duplexes on the surfaces of the spiral channel of the chip and destabilizes the MM duplexes but not the PM duplexes. The nanoparticle does not bind to the fully coiled duplex, but does only target the ssDNA segments (bubbles) of the duplex in the course of dehybridization and accelerate the propagation of the bubbles and unzipping of the duplex. This mechanism of destabilization causes a preferential removal of the MM duplexes, rather than the PM ones, and hence the signal is preserved, while the specificity is enhanced. We have also studied the influence of several governing factors of the method, evaluated the performance of the technique upon the variation of the DNA sequences, and applied the method for detection of KRAS gene SNP variations in genomic samples. Furthermore, the SNP discrimination is achieved at a single temperature, alleviating the difficulty of temperature optimization for multiple targets of different melting temperatures in multiplex analysis. In contrast to the other attempts (e.g. molecular beacons) to enhance the specificities of DNA hybridization, no complicated design for the DNA probe sequence is required and high specificity is effectively achieved via a simple wash step subsequent to DNA hybridization. This simplicity is an advantage which, together with the robustness upon sequence variation and compatibility with multiplex analyses, makes this technique a promising tool to be used in DNA hybridization-based microarrays with the potential to reduce false positive/false negative results and improve the accuracy of the microarray results.
Other than hybridization, for DNA amplification we have developed the nanoparticle-assisted helicase-dependent amplification (HDA), termed nanoHDA, by enhancing the efficiency of conventional HDA using AuNPs. The nanoHDA technique is then coupled to our AuNP-enhanced technique for detection of SNPs in the KRAS gene. To the best of our knowledge, this is the first report on the use of nanoparticles for improving an isothermal amplification technique.
We attribute low HDA signals on lane 3 to the low efficiency of helicases to denature long dsDNA templates, which may cause HDA to fail in amplifying long gDNA, but not short ssDNA. In contrast, PCR is successful (lane 5) because it denatures the template by heating, which is capable of quickly denaturing even long dsDNA. To overcome the issue of DNA length, we treated the gDNA template with the restriction enzyme NlaIII (New England Biolabs) to generate DNA fragments of reduced lengths. This restriction enzyme was chosen to perform a digestion and create a 240-bp fragment which contains the KRAS sequence. Lanes 4 and 6 show the results of improved intensities for the HDA amplicons obtained from the 240-bp restriction fragment. These results are consistent with the previous report by Tong et al. that the use of a restriction enzyme as an additive in the HDA reagents improved the amplification of a bacterial DNA [29].
These improved results in lanes 4 and 6 also confirm our hypothesis that the HDA efficiency for long DNA templates is low because of the limited capability of helicase-mediated template denaturation. However, the improved signal is still not comparable to the signal obtained from the HDA amplicons generated from the 162-gene fragment template (lane 2). As inspired from the use of nanoparticles in PCR, we added gold nanoparticles (AuNPs) in the HDA reagents to assist in the helicase-mediated denaturation of templates, a new method we dubbed nanoHDA. The hybridization signals in Lanes 8 and 9, which were obtained from HDA on gDNA with AuNP added to the amplification mixture. Even when AuNPs were used, the template should still be digested with the restriction enzyme, as seen from the low intensity in Lane 7 when only AuNP but not restriction enzymes was used. A comparison between the signals in lanes 8 and 9 shows that the use of different sized AuNPs (5 nm, 10 nm, respectively) has a similar enhancing effect on HDA. On the basis of these results, we speculate two ways that AuNPs assist helicases in dsDNA denaturation and thus enhance HDA. First, AuNPs may have a preferential affinity for ssDNAs versus dsDNAs. Thus in a similar fashion to that of single-stranded binding (SSB) protein, nanoparticles may bind to the ssDNA segments and prevent them from renaturation, which assists the helicase-dependent denaturation. Second, AuNPs may be able to directly affect the dsDNA segments and enhance their denaturation [21, 58, 59], a capability that has not been reported for SSB protein. Once bound to a partially denaturated DNA, AuNPs destabilize the neighbouring base-pairs and accelerate denaturation of the dsDNA segments.
To examine if a greater number of AuNPs enhance HDA even more, we studied the effect of different amounts of AuNPs on nanoHDA.
To confirm if HDA inhibition at high AuNPs concentration is due to a loss of HDA enzymes on the nanoparticle crusts, different enzyme mix concentrations were used and the results were compared. As shown in
NanoHDA also reduced the nonspecific amplification of primer-dimers. Their formation is evidenced in the results of the primer-dimer peaks obtained using capillary gel electrophoresis (CGE). As shown in
A comparison between the kinetics of HDA and nanoHDA was also conducted on the NBA chip to further understand the effect of nanoparticles on enhancing HDA.
A property resulted from the interaction between DNA bases and nanoparticles is used to enhance the reaction efficiency of PCR, as reported by several groups, and different mechanisms were proposed for this enhancement effect [51, 52]. First, Li et al. suggested that the preferential binding of single-stranded DNA (ssDNA) to AuNP surfaces, in a manner similar to single-strand binding protein (SSB), increased the specificity and sensitivity of PCR [53]. Second, the excellent heat-transfer property of nanoparticles is proposed to have shortened the reaction time for PCR [54], but this idea was later criticized by others [55]. Furthermore, Mi et al. suggested that AuNPs modulated the polymerase activity and enabled a hot start-like effect that suppressed nonspecific amplification at low temperature [56]. As inspired by these reports, we try to further enhance our nanoHDA technique by reducing the temperature from 65° that is used in conventional thermophilic HDA. However, we observe a significant signal reduction as the nanoHDA temperature decreases from of 65° to 40° (See
The nanoHDA technique provides an efficient platform for amplification of human genomic DNAs for subsequent hybridization-based detections. Therefore, we aim to combine nanoHDA with our AuNP-wash method to enable SNP detection using nanobioarray (NBA) chips. This combination allows for high-throughput SNP genotyping of the human genome. In the combined method, the amplicons were first prepared by a 1-h amplification using nanoHDA (10 nm AuNP, 0.3 nM) from 1 ng of gDNA with different alleles of KRAS gene. Thereafter, the HDA amplicons hybridized to an array of surface-bound oligonucleotide probes on the surface of an NBA chip, and finally the mismatched DNA duplexes were removed using the AuNP-wash method. As indicated by high sensitivity and specificity of the signals in
In conclusion, we used gold nanoparticles (AuNPs) to improve sensitivity and specificity of helicase-dependent amplification (HDA). Our results show that preferential binding of nanoparticles to ssDNA facilitates helicase-mediated DNA denaturation and hence accelerates HDA and improves amplification sensitivity. In the presence of nanoparticles, the formation of primer-dimers were also suppressed which contributed to the high specificity of the technique. Finally, we successfully demonstrated SNP detection on human gDNA samples by coupling the nanoHDA technique with the AuNP-enhanced hybridization technique.
Gold nanoparticles (with citrate and tannic acid) of 5-, 10- and 20-nm diameter were purchased from Sigma Life Science and 12-nm diameter gold nanoparticles (capped with citrate) were obtained from NanoComposix (San Diego, Calif.). Sodium dodecyl sulphate (SDS), 3-aminopropyltriethoxysilane (APTES), 25% glutaraldehyde, cetyltrimethylammonium bromide (CTAB) and Triton X-100 were purchased from Sigma-Aldrich. Negative photoresist (SU-8 50) and its developer were purchased from MicroChem Corp. (Newton, Mass.). Circular glass chips with 4-in. diameter and a 0.6-in. centre hole were obtained from Precision Glass & Optics (Santa Ana, Calif., USA).
All the reagents and materials required for surface plasmon resonance (SPR) experiments including 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), ethanolamine, HBS-N Buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl) and CMS sensor chips, were provided by GE Healthcare (UK).
All oligonucleotides (listed in Table 2) were synthesized and modified by Integrated DNA Technologies (Coralville, Iowa). Target oligonucleotides (20- or 60-mer) representing different SNPs of KRAS gene codon 12 (G12A (A) and wild-type (W)) and also 20-mer of B and NB targets (fungal pathogenic sequences [32]) were modified with a biotin molecule at the 5′-end. The probe sequences were designed in such a way that the SNP sites were located at the centre of the oligonucleotides. The 20-mer probe oligonucleotides were modified with an amine group and a C12 spacer at the 5′-end.
GGC GTA GGC AAG AGT GCC TTG ACG ATA CAG-3′
The genomic DNA samples, containing different allele compositions of the KRAS gene codon 12 were obtained from QIMR Berghofer Medical Research Institute (Brisbane, Australia). In order to obtain the 80-bp PCR products, a pair of forward and reverse primers (See Table 2) was used. A custom PCR protocol on a thermocycler (Cetus, Perkin Elmer) was used for DNA amplification. The thermocycling was initiated by 3 minutes of denaturation, followed by 30 thermal cycles of 95° C. for 40 s (denaturation), 55° C. for 30 s (annealing) and 72° C. for 60 s (extension), and terminated by 10 minutes of final extension at 72° C. The amplified products were purified using a nucleotide removal kit form Qiagen Inc. (Toronto, ON, Canada).
The CD-NBA chip comprises of a PDMS slab (4 in. diameter) with 96 radial microchannels, sealed reversibly to a circular glass chip. The width of straight radially arranged channels was 200 μm and the height was 35 μm. The probe immobilization procedure was similar to the previously reported methods [19, 64, 65]. Briefly, 0.5 μL of probe solution (in 1.0M NaCl+0.15M NaHCO3) was added to the inlet reservoirs of the CD-NBA chip, and it was placed on a rotating platform. The solutions were introduced into the radial channels by spinning the circular chip at 400 rpm for 3 min. The probe solutions were driven out from the channel after 20 min. of incubation at room temperature by spinning the chip at 1800 rpm for 1 min. Subsequently, the radial PDMS slab was peeled off, leaving behind 96 radial probe lines printed on the glass chip, which was then rinsed and dried. Thereafter, another PDMS slab with 96 spiral channels was sealed against the glass chip pre-printed with the probe lines to carry out the DNA hybridization. The target solution (1 μL), prepared in hybridization buffer (1×SSC+0.2% SDS) with a final concentration of 10 nM, was added to the inlet reservoir and then flowed in the spiral channel (100 μm wide) using a spin rate of 900 rpm. This spin rate resulted in ˜13 min. of dynamic hybridization of the targets to the complementary probes at the intersections of spiral channels with the radially arrayed probe lines. High-temperature experiments were achieved by heating the CD-NBA chip using a hot air blower. The temperature was calibrated in a separate experiment using a temperature sensor placed on the glass chip surface, sealed with the PDMS slab to it.
The washing procedure was performed after DNA hybridization. The wash solution was SSC with NaCl concentrations that range from 10 to 300 mM. The washing buffer contained either no AuNPs or AuNPs of various concentrations from 0.2 to 40 nM. In order to stabilize the AuNPs against salt aggregation, they were loaded with DNA oligonucleotides, with sequences irrelevant to the target strands, prior to addition to the wash buffer. This was performed by mixing various concentrations of 12- or 20-mer oligonucleotide with AuNPs and incubating the mix at 95° C. for 5 min. Afterwards, 2 μL of the AuNP wash buffer was added to the inlet reservoirs of the spiral channels. Dynamic wash was performed by spinning the CD-NBA chip at spinning rates of 700 to 1500 rpm. Stop-flow wash was performed by spinning the chip at 2200 rpm for 20 s in order to fill the channels with the wash buffer, incubating for 15 min. (stop-flow), and then ejecting the buffer with a spin rate at 2200 rpm for another 20 s. After washing (dynamic or stop-flow) was completed, streptavidin-Cy5 solution (50 μg/ml in 1×PBS buffer) was added to the inlet reservoir and allowed to flow in the channel by spinning at 1500 rpm. Finally, the spiral PDMS slab was peeled off from the glass chip.
The fluorescence detection was carried out by scanning the glass chip on a confocal laser fluorescent scanner (Typhoon 9410, GE Healthcare) at 10 μm resolution, as previously described [20, 22]. The excitation and emission wavelengths were 633 and 670 nm, respectively. The photomultiplier tube voltage was set at 600 V. The scanned image was analysed by IMAGEQUANT 5.2 software.
In order to study the kinetics of adsorption of DNA oligonucleotides on the surface of AuNPs, the fluorescence quenching measurement was conducted. Cy5-labeled W20 oligonucleotides (8 nM) were prepared in 1 ml of sodium citrate buffer (15 mM) in a polystyrene cuvette. The buffer contained NaCl concentrations of 0, 10, 30, 50, 70, 90, 110, 130 and 150 mM. The cuvette was placed in the holder of a spectrofluorometer (Photon Technology International). Thereafter, 1 ml of aqueous AuNP colloid (80 nM) was added to the cuvette and the content was mixed. Immediately after, the fluorescence intensity (excitation at 650 nm and emission at 670 nm) was monitored for 7 min. using the time-based mode.
The SPR measurements were performed on BIAcore X100 (GE Healthcare) as previously reported [21]. Briefly, the immobilisation of the amine-labelled 20-mer probes (A) was performed on the surface of a sensor chip (CMS), using a company-developed method [21, 33]. The carboxylic groups on the sensor surface were activated by an EDC/NHS mixture (1:1 v/v). Then, the amine-labeled probe molecules were immobilized on the sensor surface by running the immobilization solution containing the probe molecules (50 μM) and CTAB (0.6 mM) over the sensor surface. Finally, unreacted succinimide groups were deactivated using an ethanolamine solution (pH 8.5). The target solutions were prepared in the HBS-N buffer with DNA target concentrations of 10, 20, 40, 80 and 160 nM. The rate constants of DNA hybridization and dehybridization were determined using the multi-cycle kinetic procedure. Briefly, 10 nM target solutions were first continuously flowed for 60 s over the sensor chip surface (with immobilized probe). After hybridization, washing was achieved by a continuous flow of wash buffer over the sensor surface for 240 s. In the stringent wash experiment, the HBS-N buffer was used as the wash buffer. However, the AuNP wash buffer contain 5-nm AuNPs (10 nM) in the HBS-N buffer. The nanoparticles in the AuNP wash buffer had been previously loaded with the 20-mer oligonucleotides (stabilizers with a sequence unrelated to the target and probe), by mixing the stabilizers with AuNPs in water and then incubating the mix at 95° C. for 5 min. After each hybridization-wash cycle, the sensor surface was regenerated (all the target strands were washed away) by running an alkaline wash (50 mM NaOH) for 30 s. This cycle of hybridization, wash and regeneration was repeated for the other 4 target concentrations of 20, 40, 80, 160 nM.
Helicase-Dependent Amplification (HDA) and nanoHDA
All probe oligonucleotides, primers and gBlocks Gene Fragments (listed in Tables 2 and 3) were synthesized and modified by Integrated DNA Technologies (Coralville, Iowa). In order to obtain the 92-bp PCR products, a pair of forward and reverse primers (See Table 3) was used.
All the reagents for HDA and also the b-DNA template were purchased from Biohelix Corporation (Beverly, Mass., USA). Taq DNA polymerase and PCR dNTP mix were purchased from Thermo Fisher Scientific (Waltham, Mass.). The restriction enzyme NlaIII was purchased from New England Biolabs (Ipswich, Mass.).
To setup a 25 μL of 1×HDA reaction, 2.5 μL of 10× annealing buffer, 0.75 μL of 100 mM MgSO4, 2.5 μL of 500 mM NaCl, 1.75 μL of IsoAmp dNTP solution (200 μM dNTPs, 3 mM dATP), 1 μL of IsoAmp III enzyme mix 1 ng of DNA template, 0.75 μL of forward and reverse primer (2.5 μM), and 12.75 μL ddH2O were pipetted into a 0.2 mL centrifuge tube. The enzyme mix consisted of 10 U of polymerase (an analog of Bst that doesn't have 3′-5′ exonuclease activity), 50 ng of helicase (Tte-UvrD), 200 ng Tte-MutL (a cofactor of helicase that stimulates and enhances the unwinding performance), and 25 ng ET-SSB, The HDA reaction mixture was briefly vortexed, followed by 30 s centrifugation at 1500 g. The HDA reaction mixture was then overlayed with 50 μL of silicone oil and incubated for 120 min (unless otherwise noted) in a water bath at 65° C. For nanoHDA experiments different amounts of AuNPs were added to the forward primer solution and the mix was kept overnight before being added to the HDA mix. The amplified products were purified using a nucleotide removal kit (Qiagen, Hilden, Germany).
PCR amplification was performed on a thermocycler (Cetus, Perkin Elmer), as previously described [19]. To setup a 50 μL of 1× reaction, 5 μL of 10×PCR buffer, 3 μL of 50 mM MgCl2, 5 μL of dNTP mix (2 mM each dNTP), 0.5 μL Taq DNA polymerase solution (1.25 U), 1 ng of DNA template, 8 μL of forward and reverse primer (2.5 μM), and 19.5 μL ddH2O were pipetted in a 0.2 mL centrifuge tube, mixed by a brief vortex followed by 30 s centrifugation at 1500 g. The thermocycling was initiated by 3 min of denaturation, followed by 30 thermal cycles of 94° C. for 40 s (denaturation), 65° C. for 30 s (annealing) and 72° C. for 60 s (extension), and terminated by 10 min of final extension at 72° C. The amplified products were purified using a nucleotide removal kit (Qiagen).
All the CGE experiments were performed on Bioanalyzer 2100 (Agilent Technologies, Santa Clara, Calif.). A DNA 1000 kit was used to analyze the 92-bp amplicons. Briefly, 1 μL of purified amplicons were diluted 1:10 in ddH2O and added together with 5 μL of marker solution (low and higher markers) to the DNA chips. The electropherograms were obtained using the 2100 Expert software (Agilent Technologies). The chips may be cleaned and re-used, as previously described [66].
It is understood that the examples in the foregoing disclosure in no way serve to limit the scope of this invention, but rather are presented for illustrative purposes. As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the scope thereof.
This invention has a wide range of aspects. Without limitation, the aspects include each of the following:
1. A nucleic acid hybridization method, comprising:
This application claims priority to U.S. Provisional Application No. 62/144,827, filed Apr. 8, 2015. The content of the priority application is incorporated by reference in its entirety.
