Combined solution phase and solid phase DNA amplification

Abstract

To provide combined solution phase and solid phase DNA amplification.SOLUTION: Described is means for efficiently amplifying and detecting certain nucleic acid sequences from a population. The selected population can be further characterized, for example by sequencing. The method involves combined solution phase and solid phase amplification.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for sequence-specific amplification of a subpopulation of nucleic acid fragments from a broader population of nucleic acid sequences. The present invention accurately detects only the desired target sequence. Aspects of the present invention relate to nucleic acid constructs for use in such methods. A means for efficient targeted amplification and detection of specific nucleic acid sequences from a population is described. [Background technology]

[0002] Identification of pathogens in blood and other biological samples is critical for the effective treatment of many disease conditions, including sepsis. Sepsis causes five deaths every hour in the UK, and 25% of all sepsis survivors suffer from permanent, life-changing sequelae. Early detection and identification of the causative agent of bacterial infection is essential to prevent the development of sepsis and for successful treatment. Current methods of detection and identification of blood-borne infections include the use of blood cultures and antibiotic susceptibility assays. These methods involve culturing cells, which is both time consuming and expensive. Often, septic shock occurs before cell culture results can be obtained.

[0003] The use of current molecular detection methods such as PCR, which can identify pathogens from samples by analyzing pathogen-derived nucleic acids, is limited by a low overall sensitivity level. This is particularly problematic when analyzing human samples, due to the presence of large amounts of non-target (human) nucleic acids. These non-target nucleic acids can either inhibit downstream purification and amplification of the target (pathogen) nucleic acid, resulting in false-negative results, or give false-positive readings of pathogen nucleic acid due to non-specific amplification.

[0004] For example, there are some antibiotic resistance genes that have multiple variants due to point mutations or other polymorphisms, and the presence of a certain mutation may lead to different treatment strategies compared to another mutation.Therefore, it is important that the identification of these polymorphisms is performed in a timely and cost-effective manner.Current identification strategies using PCR and other amplification reactions have limited capacity, and laboratories can use sequencing to elucidate internal sequences and identify polymorphisms, but this is time-consuming and expensive to carry out.Therefore, selective amplification of a population of target nucleic acids compared to non-target nucleic acids is of great value to the medical industry and research.

[0005] Amplification reactions generally rely on a pair of amplification primers for each desired sequence to be amplified. Although several pairs of non-immobilized primers can be combined, due to cross-hybridization between primers, the combination of a large number of primer pairs is generally not feasible. Therefore, multiplex PCR is generally performed using separate primers in separate solution volumes, requiring large amounts of sample. The present invention describes an improvement in amplification reactions that allows sequence-selective amplification of multiple different primers in a single sample volume.

[0006] A method for emulsion PCR is also disclosed in which individual templates are diluted so that the water bubbles in the oil emulsion contain less than one template molecule per bubble on average. A bead with a single immobilized primer can be included, and a second primer is in solution. Thus, amplification is carried out using a mixture of two primers, one of which is immobilized and the other is in solution. However, this gives only a single amplification product per reaction volume, in contrast to the method described herein, in which the first amplification product is further amplified.

[0007] Amplification methods are also known in which both primers are immobilized, such as Illumina's cluster-based bridge amplification, in which case there are no primers in free solution. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a two-phase amplification reaction, including both solid-phase and liquid-phase (thermocycling or isothermal) amplification reactions, that can simultaneously generate DNA amplicons in solution and on a surface using combinations of immobilized and non-immobilized primers and target DNA templates. The presence or absence of amplicons can be detected in a post-amplification end-point assay or in real time. This two-phase approach differs from the current state of the art, including emulsion PCR, because it includes both solid-state, i.e., surface-based, and liquid-phase amplification.

[0009] The nucleic acid sample can be amplified using one or more non-immobilized primers. This liquid-phase generated DNA, and the target DNA template, can also or subsequently serve as a template for further reactions in the solid phase, whereby one or more immobilized primers interact with the liquid-phase template as part of the DNA amplification reaction. By immobilizing primers of different sequences at known locations and using a detection system that can identify the locations (e.g., using ISFETs on CMOS IC chips or array-based fluorescent / optical imaging systems), the readout provides additional discrimination power to the system.

[0010] Here we propose a novel method for achieving multiplex sequence-specific amplification of subpopulations of nucleic acid fragments from a broader population of nucleic acid sequences.

[0011] Methods are described, including methods for the amplification and analysis of nucleic acid sequences, including: a. taking a system having a liquid volume in contact with a solid support, the liquid volume comprising a nucleic acid sample, two or more non-immobilized amplification primers, and reagents for nucleic acid amplification, the solid support having one or more immobilized amplification primers; b. performing a nucleic acid amplification reaction using a non-immobilized amplification primer to generate a first non-immobilized nucleic acid amplification product; c. further amplifying the first non-immobilized nucleic acid amplification product using one or more immobilized amplification primers to generate an immobilized second nucleic acid amplification product; di examining in real time the localized signal generated at the location of the immobilized second nucleic acid amplification product; or ii. removing the liquid volume and the first non-immobilized amplification product and replacing it with a new liquid volume having a primer that hybridizes to the immobilized second nucleic acid amplification product; Either of the following, and 1. Direct detection of hybridized primers, or 2. Flowing a solution containing at least one nucleotide over the immobilized second amplification product to extend the hybridized primer. Any of the following, and e. Detecting the presence, absence or sequence of a second nucleic acid amplification product.

[0012] Methods can be performed in which non-immobilized amplification primers in a liquid volume are released from the solid phase prior to amplification. Nucleic acid amplification reactions using non-immobilized primers can use one or more pairs of non-immobilized primers. Any stage of nucleic acid amplification can be isothermal or can be performed by thermal cycling.

[0013] According to this method, the primer hybridized to the second nucleic acid amplification product can be fluorescently labeled and the subsequent measurement of fluorescence can be achieved. Alternatively, the detection of the amplification product can be performed using the detection of protons released from the incorporation of nucleotides, for example, via an ISFET sensor.

[0014] The method can be performed such that the first and second amplification products are obtained using amplification primers having the same nucleic acid sequence.

[0015] The method may further include an immobilized amplification primer that amplifies an internal portion of the first amplification product such that the second amplification product is shorter than the first amplification product.

[0016] The method can further include exposing the same first liquid volume to two or more immobilized amplification primers, each primer amplifying a different sequence. The two or more immobilized amplification primers can differ by a single base, thereby detecting single base variants in the first nucleic acid amplification process.

[0017] The immobilized primers of this method can be present on an open microarray or in separate wells. Separate wells can be exposed to the same reagent volume simultaneously. If the wells contain immobilized primers of different sequences, different amplicons can be generated in different wells, thus allowing multiplex amplification in a single liquid volume.

[0018] A still further aspect of the invention provides a system for amplification and detection of nucleic acid sequences comprising a liquid volume in contact with a solid support, the liquid volume comprising a nucleic acid sample, two or more non-immobilized amplification primers and reagents for nucleic acid amplification, the solid support having one or more immobilized amplification primers, and the system comprising a sensor for detecting an amplification product.

[0019] The amplification products can be detected optically or electrically. Optical detection requires that the amplification products be labeled with a fluorescent marker, which can then be detected by an optical regime.

[0020] Whether optical or electrical output is detected, sensing can be performed via an array of silicon-based sensors, such as field effect transistors (FETs). The FETs selected can be any suitable CMOS chip, including but not limited to ISFETs. The sensors are configured to integrate the sensed signal in time, or in space, or both, to identify the presence, absence, or possibly quantum of amplification product present. Unlike some deployments of optical arrays, they are not configured to image the output. [Brief description of the drawings]

[0021] [Figure 1] Schematic diagram showing single localized anchored primer amplification and detection (LAPAD). [Diagram 2] Schematic diagram showing multiple localized anchored primer amplification and detection (LAPAD). [Diagram 3] Schematic showing a run-off run. It is performed in an open system requiring on-chip flow capabilities. During the amplification reaction, product amplicons from the liquid phase land on the solid-phase primers and are extended to form a forest of extended amplicons on the chip surface. After amplification, the amplification products and reagents are washed away leaving single-stranded amplicons on the chip surface. Run-off primers and enzymes are loaded onto the chip and after a short hybridization step, all four nucleotides are flowed in together, creating a burst of protons on those ISFETs with the extended oligos. This proton burst is seen very clearly with minimal data processing. [Figure 4] Figure 4 shows the run-off workflow and data therefrom. The first row represents the amplification stage of the assay, generating clusters of extended amplicons on the chip surface. This is followed by a dehybridization step, e.g., using heat, to generate single-stranded amplicons on the chip surface with no product in the liquid phase. Following hybridization of the run-off primer with the enzyme, all four nucleotides flow across the chip and a signal is generated on the specifically extended ISFET. [Diagram 5] Figure 5 shows sample ISFET data showing the runoff signal generated after PCR for three target templates. Data shown are for E. coli (80 base product, therefore a maximum runoff of 60 bases), Enterococcus faecalis, and Serratia marcescens after specific amplification of the relevant targets. Data has not been modified and all ISFETs are shown. [Figure 6]Figure 6 shows a multiplexed chip spotted with three anchored primers and a positive template control. PCR with Ef template followed by run-off. The heat map shows the ISFET voltage measured after approximately 15 seconds of nucleotide flow. The ISFETs are seen to match the spotting pattern and correspond to both Ef and the positive control. Traces on the right show examples of ISFET responses for each ISFET group. [Figure 7] Figure 7 shows a sequencing run. Instead of all four nucleotides being added for run-off, the nucleotides are added sequentially. It is performed in an open system requiring flow capabilities on the chip. During the amplification reaction, product amplicons from the liquid phase land on the solid phase primers and are extended to form a forest of extended amplicons on the chip surface. After amplification, the amplification products and reagents are washed away leaving single-stranded amplicons on the chip surface. Sequencing primers and enzymes are loaded onto the chip and after a short hybridization step, all four nucleotides are individually flowed in, creating a burst of protons on the ISFET with the amplification product and the correct template base. Single base resolution can be achieved. [Figure 8] Figure 8 shows the sequencing workflow and data therefrom. The first row represents the amplification stage of the assay, generating clusters of extended amplicons on the chip surface. This is followed by a dehybridization step, e.g., using heat, to generate single-stranded amplicons on the chip surface with no product in the liquid phase. Following hybridization of the run-off primer with the enzyme, all four nucleotides flow across the chip individually, generating a signal on the ISFET that corresponds to where the nucleotide was incorporated and an amplification product was generated. [Figure 9]Figure 9 shows the real-time detection workflow. It can be performed on a platform with no or minimal fluid flow after the chip amplification. The reaction can be performed in one or more permanently sealed reaction chambers. During the amplification reaction, the product amplicons from the liquid phase land on solid-phase oligos, modifying and extending the specific oligos. In a low-buffered, closed system, protons are generated near the solid phase during the extension stage of each cycle of the amplification reaction. Pushing the asymmetry to give a significant excess of forward primer in the liquid phase effectively generates a "mini-runoff" burst of protons in each cycle, which should be detectable in later cycles. As an alternative to primer asymmetry at the beginning of the amplification reaction, additional primers can be released or added during the amplification reaction or at set times. The amplification reaction is performed in low buffer to allow real-time detection of the pH signal. [Figure 10] Figure 10 shows data from real-time detection: chip with multiple anchored primers spotted in the block, PCR (3:1 asymmetric) with a 6-chamber gasket (three chambers fluid-tight), run-off on a flow station with a single chamber. The liquid phase shows a real-time pH signal indicating the reaction was progressing. As a confirmation of amplification, after PCR, the chip was probed with a fluorescent label targeting the distal end of the amplicon, indicating that the anchored primer was extended during the PCR reaction. [Figure 11] 1 shows the average delta signal from the ISFET of Example 1. [Figure 12] FIG. 12 shows the delta signal from all ISFETs for the anchored primer target positives of Example 1. [Figure 13] FIG. 13 shows the delta signal from all ISFETs for the anchored primer target negative of Example 1. [Figure 14] FIG. 14 shows the delta signal from all ISFETs for the anchored primer control of Example 1. [Figure 15] FIG. 15 shows the average ISFET signal results for the positive, negative and control primers of Example 1. [Figure 16] FIG. 16 is a gel electrophoresis image showing that solution-phase amplicons were generated following PCR of Serratia marcescens in Example 1. [Figure 17] FIG. 17 shows the Sensospot fluorescence image of Example 1. [Figure 18] FIG. 18 shows the average delta signal from the ISFET of Example 2. [Figure 19] FIG. 19 shows the delta signal for anchored primer targets 1-4 positive in Example 2. [Figure 20] FIG. 20 shows the delta signal from all ISFETs for the anchored primer negative of Example 2. [Figure 21] FIG. 21 shows the delta signal from all ISFETs for the anchored primer control of Example 2. [Figure 22] FIG. 22 shows the average ISFET signal results for the positive, negative and control of Example 2. [Diagram 23] FIG. 23 is a gel electrophoresis image of Example 2. [Figure 24] FIG. 24 is a Sensospot fluorescence image of Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] A system with the ability to generate different sequence readouts from the same reaction volume is described.

[0023] Briefly, the system includes a two-phase amplification reaction in which a liquid-phase (PCR or isothermal) amplification reaction occurs in the presence of immobilized primers on a solid phase, which further participates in the generation of an amplification reaction product. The liquid-phase (PCR or isothermal) amplification reaction can use one or more primers and a target nucleic acid template to generate a nucleic acid amplification product in solution, and when sufficient product is generated, a corresponding signal is detected by the system. This liquid-phase generated nucleic acid amplification product and the target nucleic acid template can also or subsequently function as a template for further reactions on the solid phase, whereby one or more immobilized primers interact with the liquid-phase template as part of the amplification reaction, and subsequently generate a nucleic acid sequence detectable by the system. By immobilizing the primers at known locations and using a detection system that can identify the location (e.g., using an ISFET on a CMOS IC chip or an array-type optical imaging system), the readout allows for selective amplification and detection of many amplicons in the same device.

[0024] The amplification reaction is carried out in liquid phase in the presence of one or more additional primers immobilized on a solid phase. The immobilized primers can be of the same sequence as the one or more liquid phase primers or correspond to an internal sequence of the nucleic acid template generated in liquid phase. The former allows a second call to be made from the same reaction components, the latter allows a second call to provide additional confirmation and increased confidence that the correct amplification reaction product has been generated and / or provide further information about the internal sequence of the target template.

[0025] The detection mode described herein can use ISFET CMOS technology, but is also applicable to existing nucleic acid amplification reactions and microarray methods, as well as customized systems for integrating real-time detection of both liquid-phase and solid-phase reactions. The redundancy potential of ISFET CMOS allows for a significant number of replicates for each assay and multiple target genes for each target, thereby improving the reliability of the system by relying on multiple readouts for each target. This design also facilitates the incorporation of positive and negative controls into each reaction vessel, ensuring that the correct call is made when the controls function as required.

[0026] Below are various embodiments of combined liquid and solid phase amplification reactions.

[0027] DNA amplification readout The immobilized primers described may be the same as or similar to one or more solution-phase primers and participate in the amplification reaction such that an increased signal is generated from the immobilized primers when compared to the amplification performed in the absence of solution primers, thereby adding confidence to the result, but without adding any additional information to the sequence generated. For example, asymmetric PCR in liquid phase can be compensated by having the primers depleted in the liquid phase present on the solid phase. As the amplification reaction proceeds, the liquid phase products generated are prompted to interact with the solid phase, generating both liquid phase and solid phase amplicons. The immobilized amplicons can be detected. The presence of this readout across multiple locations where primers are immobilized increases confidence that amplified material is indeed present in the sample at that time. If immobilized primers with different sequences do not produce a positive result, the confidence of the result is increased.

[0028] DNA Amplification with Internal Verification The immobilized primers described can correspond to internal sequences of the DNA template generated in the solution phase, so the signal generated in the solid phase is a clear confirmation that the correct product was generated in the solution phase. This confirmation confers a significant advantage over standard confirmation tests such as melting curve analysis and electrophoretic gel imaging in that it demonstrates that the solution phase amplicons are not generated by primer dimers, mismatched primers, or other non-specific reactants (e.g., signals generated by interacting with host (human) DNA if the intended target is a pathogen in the host sample), and further sequence information is inferred in the readout. The internal validation signal is similar to standard laboratory validation procedures such as melting curve analysis, electrophoretic gel imaging, etc.

[0029] The use of immobilized primers corresponding to internal sequences in the target template allows for additional specificity to be imparted to the system, such that the combination of liquid and solid phase reactions identifies more sequence information than a liquid phase reaction by itself, thereby improving the reliability of the results compared to liquid phase alone.

[0030] DNA amplification using internal sequence information In addition to the confirmation steps already identified, the possibility of having multiple immobilized primers corresponding to different regions of the solution-phase amplification product is also available, so that one or more can be used to provide internal information in addition to simply confirming that the correct product was made in solution phase.

[0031] This can take the simple form of multiple internal primers from the amplification product, each adding credibility to the inferred results by providing multiple sequence alignments, or in more advanced versions, can be used to identify internal mutations within the target gene that correspond to point mutations and other polymorphisms. For example, there are some antibiotic resistance genes that have multiple variants due to point mutations or other polymorphisms, and the presence of a particular mutation may result in a different treatment strategy compared to another mutation. It is therefore important that the identification of these polymorphisms is done in a timely and cost-effective manner. Current identification strategies using PCR and other amplification reactions have limited capacity, and laboratories can use sequencing to elucidate the internal sequence and identify the polymorphisms, but this is time-consuming and expensive to perform and is often not done.

[0032] In this embodiment, a single primer pair can be used in liquid phase to amplify all variants of a gene, and one or more immobilized primers corresponding to point mutations or polymorphisms can be used in solid phase to identify one or more variants. Each combination of internal immobilized primers can provide the user with a wealth of information about the overall sequence of the amplified gene, making clinical decisions easier and faster to make.

[0033] Runoff (including multiple runoffs) Amplification reactions are generally carried out in buffers of high ionic strength. In sequencing systems that measure the proton release of incorporated nucleotides, the amplification products can be detected by replacing the amplification buffer with a buffer of low buffer capacity. Alternatively, the amplification products can be detected using fluorescently labeled primers or through the use of fluorescently labeled nucleotides.

[0034] Runoff is performed in an open system that requires flow capabilities on the chip. During the amplification reaction, product amplicons from the liquid phase land on solid phase primers and are extended to form a forest of extended amplicons on the chip surface. After amplification, the amplification products and reagents are washed away leaving single-stranded amplicons on the chip surface. Runoff primers and enzymes are loaded onto the chip and after a short hybridization step, all four nucleotides are flowed in together, creating a burst of protons on those ISFETs with the extended oligos. This proton burst is seen very clearly with minimal data processing.

[0035] To perform electron / proton detection, at the end of the aforementioned amplification steps in both solution and solid-phase reactions, the liquid phase components are washed out of the cartridge and replaced with a solution of low buffer capacity. Primers and enzymes are then followed, either in sequence or in combination, followed by the flow of nucleotides, which upon incorporation releases protons and generates a corresponding signal on the detection platform, e.g., mV change of the associated ISFET. The primers used for run-off can correspond to any position in the amplified template, including generic sequences added during the previous amplification reaction. The use of amplicon-specific primers inside the solution amplification product increases the reliability of the results by confirming that the correct material was made in the solid phase, but may increase the complexity of the system by allowing higher multiplexing. If generic sequences are added in the previous amplification reaction, it is possible to limit the scale of solution-phase multiplexing, thereby improving the efficiency of the reaction, but this may limit the reliability of the readout due to the possibility of misfiring of the generic sequences.

[0036] By knowing the location of a particular immobilized primer, the identity of the target can be determined when protons generated during the run-off reaction result in a signal at the particular ISFET sensor to which the particular primer is immobilized. In this embodiment, multiple replicates are possible for each assay target, with the scale of multiplexing limited by spatial separation, the number of wells and sensors per device, and bioinformatics challenges.

[0037] The system can utilize a single runoff reaction or multiple runoff reactions, with or without a dehybridization step between each run. Additional runoffs can be used to check prior signals so that confidence in the internal sequence can be increased. For example, if a generic sequence was used in a prior amplification reaction, the first runoff can use that generic sequence to identify which assays generated the signal, and subsequent runoffs can use specific primers corresponding to those specific assays to confirm that the correct products were made.

[0038] Primers of different sequences can be immobilized at different locations on a solid support so that multiple target sequences can be analyzed in parallel.

[0039] Sequence information (single or multiple bases at a time) At the end of the aforementioned amplification step, the liquid phase components can be washed out of the cartridge and replaced with a low-buffer solution, similar to the run-off workflow. This is then followed by primers and enzymes, either in sequence or in combination, followed by the flow of individual nucleotides or combinations of two or more nucleotides, with the release of protons occurring when the correct complementary nucleotide flows across the chip and is incorporated. This workflow provides the most information-rich of all the embodiments in that it does not rely solely on the alignment of the primer with its corresponding template, but rather gives sequence information. This workflow not only confirms that the correct alignment has been performed and the correct product has been created on the solid phase, but also indicates the internal sequence, either by inference or direct identification. Similar approaches as discussed in the run-off embodiment can be used here, with multiple runs possible to provide further information, and workflows combining this approach with run-off of one or more nucleotides used are also possible.

[0040] Real-time detection By measuring the extension of the immobilized primer, the amplification process can be detected in real time. The reaction can be carried out in one or more permanently sealed reaction chambers. During the amplification reaction, the product amplicons from the liquid phase land on the solid phase oligos, modifying and extending the specific oligos. In a low-buffered closed system, protons are generated near the solid phase during the extension phase of each cycle of the amplification reaction, and can be detected.

[0041] Amplification can be asymmetric, where the two primers are present in solution at different concentrations and have no complementary primers to extend against, resulting in amplification products that are mostly single-stranded. Pushing the asymmetry to provide a significant excess of forward primer in the liquid phase effectively creates a "mini run-off" burst of protons in each cycle, which should be detectable in later cycles. As an alternative to primer asymmetry at the beginning of the amplification reaction, additional primers can be released or added during the amplification reaction or at set times. The amplification reaction is carried out in a low buffer to allow real-time detection of the pH signal.

[0042] The source nucleic acid may be a genomic polynucleotide. The source material may be eukaryotic, prokaryotic, or archaeal. One or more source materials may be provided. The source nucleic acid may be a fragment of a genome, such as a single chromosome or a single genomic locus (e.g., for rapid sequencing of allelic polymorphisms). In certain instances, the amplification may be specific to a pathogenic agent within the sample. For example, the amplification may select for bacterial or viral nucleic acids present within a human sample. The template may be DNA, RNA, or a cDNA copy thereof.

[0043] The biological material may be amplified in solution before undergoing the claimed amplification reaction. Thus, the material may have already undergone a step of sequence-based amplification before the present invention is practiced. Alternatively, the sample may be treated to add a common universal sequence to one or both ends of all strands in the sample. The universal sequence may be used as a universal sequence to hybridize to a common primer that may not exist in nature.

[0044] The invention described herein can allow for four or more levels of amplification specificity. The first amplification can be performed in solution. The second amplification can be performed using a non-immobilized primer. The third amplification can be performed using an immobilized primer of a different sequence than the second amplification primer. The presence of the amplicon can be detected using a primer specific to the amplicon generated by the immobilized primer. Thus, four levels of amplification specificity can be used, ensuring that the final amplicon is only detected if the original sequence is absolutely certain to be present in the sample. Thus, false positive results from the detection of closely related but undesired sequences can be eliminated.

[0045] The immobilization of nucleic acid on the surface is conventional. Any method of covalent or non-covalent immobilization can be used. The immobilization is ideally stable to multiple cycles of thermal cycling up to a temperature that causes separation of the nucleic acid strands. Particular methods of immobilization include UV-induced cross-linking or immobilization by forming amide or phosphorothioate bonds. EXAMPLES

[0046] Experimental data was collected for single-localized anchored primer amplification and detection (LAPAD) of Serratia marcescens (distributor: NCTC, catalog number 27137) at a single target region and multiple-localized anchored primer amplification and detection (LAPAD) of Schizosaccharomyces pombe (distributor: ATCC, catalog number 26189) at multiple target regions.

[0047] Schematics of both single and multiple LAPADs are shown in FIGS.

[0048] Example 1: Single Localized Anchored Primer Amplification and Detection (LAPAD) Amplification and detection of Serratia marcescens at a single target region. The target region at the 3' end of the amplified template DNA was hybridized to a complementary anchored primer target. During the amplification process, this anchored primer target was extended and in the next step, the same target region was detected during the extension. The primer / oligonucleotide sequences used are shown in Table 1. [Table 1]

[0049] Example 2: Multiple Localized Anchored Primer Amplification and Detection (LAPAD) Amplification and detection of Schizosaccharomyces pombe at multiple target regions. Target region 1, which is a few nucleotide bases behind the 3' end of the amplified template DNA, hybridized to the complementary anchored primer target 1. Target region 2, which is a few bases behind target region 1 and towards the 5' end of the amplified template DNA, hybridized to the complementary anchored primer target 2. Target region 3, which is a few bases behind target region 2 and towards the 5' end of the amplified template DNA, hybridized to the complementary anchored primer target 3. Target region 4, which is a few bases behind target region 3 and towards the 5' end of the amplified template DNA, hybridized to the complementary anchored primer target 4. During the amplification process, these four anchored primer targets with hybridized DNA templates were extended and in the next step, the same target regions were detected during the extension. The primer / oligonucleotide sequences used are shown in Table 1.

[0050] Experimental Method The chip platform, incorporating a "004" Ta2O5 CMOS chip, consisting of ISFETs, encapsulated in a SU-8 well, was primed using UV-based cross-linking surface chemistry. A manifold was attached to the chip surface to contain the fluidics for reactions to occur on the chip surface.

[0051] PCR was performed to generate DNA for detection, the PCR reagent mix, final volume 50 μl, is shown in Table 2. The thermal cycling conditions used are shown in Table 3. [Table 2] [Table 3]

[0052] After PCR, the chip was washed with wash buffer (0.06× saline-sodium citrate, 0.06% Tween 20) and the DNA was subsequently chemically dehybridized with 20 mM NaOH for 5 min and then washed again with wash buffer.

[0053] Next, the runoff primer mix (sequence shown in Table 1) was added to the chip surface. 3.33 μM fluorescently labeled (Cy3) primer was added in annealing buffer (5 mM magnesium acetate, 150 mM sodium chloride, 20 mM Tris pH 7.5, 0.01% Tween 20). The chip containing the runoff primer mix was then heated at 95 °C for 2 min, followed by 58 °C for 5 min and incubated at room temperature for 15 min. The chip was then washed with enzyme loading buffer (1x Thermopol®, 0.06% Tween 20).

[0054] An enzyme mixture was then added to the surface of the chip, which was made by adding 25 units / μL of custom-made Bst Large Fragment DNA polymerase to the enzyme loading buffer, and the chip was then incubated at room temperature for 5 minutes.

[0055] Incorporation of deoxyribonucleotide triphosphates (dNTPs) was achieved by using a custom-made flow system that allows dNTPs to flow over the chip surface. 12.5 μM each of deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate were added to non-carbonated RMD buffer (10 mM magnesium chloride, 25 mM sodium chloride, 0.025% Tergitol). The pH of the dNTP mixture was adjusted to pH 8 and maintained by keeping the fluid under nitrogen gas.

[0056] ISFET sensor detection The raw ISFET sensor voltage output data and the anchored primer spotting map were processed together by a custom data analysis pipeline (Galaxy Runoff pipeline v1.3.15). The voltage signal peak height from the ISFET was detected and the anchored primer ISFET voltage signal peak height was subtracted from the peak height of the adjacent upstream blank ISFET. This is done to remove system noise, including noise from the fluid flow that may mask detection. This delta ISFET sensor voltage output from all particular anchored primer sensors was averaged to determine signal detection.

[0057] E-gel electrophoresis analysis A pre-made E-gel (48-well 2% agarose gel stained with ethidium bromide) was used for the qualitative analysis of the liquid-phase PCR. Samples were collected from the chip surface after PCR, and 1 μL of sample was added to 1× E-gel loading dye before loading into the E-gel wells. A ladder was also loaded for amplicon size comparison. The gel was run for 10 min on the E-gel base, which provides an electric field for the DNA amplicons to migrate according to their size.

[0058] Sensospot Fluorescence Imaging After the run, the chip was scanned with a Sensospot fluorescent scanner under green light to illuminate the Cy3 run-off primers that would have hybridized to the amplicons generated on-chip. Based on the fluorescence intensity, on-chip amplification can be qualitatively determined.

[0059] result Detection of both assays was established quantitatively by the delta ISFET sensor voltage output. Liquid-phase PCR amplification was assessed qualitatively by E-gel analysis, and chip surface amplification was assessed by Sensospot fluorescent imaging.

[0060] Example 1: Single Localized Anchored Primer Amplification and Detection (LAPAD) Target: Serratia marcescens Chip ID: NM-248-0214

[0061] The anchored primer sequences for Example 1 are shown in Table 4. [Table 4]

[0062] The average delta signal from the ISFET is shown in FIG.

[0063] The delta signals from all ISFETs for the anchored primer target positives are shown in FIG.

[0064] The delta signals from all ISFETs for the anchored primer negative targets are shown in FIG.

[0065] The delta signals from all ISFETs for the anchored primer control are shown in FIG.

[0066] The results of the average ISFET signals for the positive, negative and control primers are shown in Figure 15. An average ISFET signal of 36.2 dmV was detected for the positive anchored primer NH2_iSp18_SmR, which was even greater than the average ISFET signal of 23.4 dmV for the control anchored primer NH2_ddl_5'+T15.

[0067] As seen in the gel electrophoresis image, solution-phase amplicons were generated after PCR for Serratia marcescens (Figure 16). Along with the control anchored primer, the generated on-chip amplicons can also be seen for the positive anchored primer NH2-iSp18_SmR in the Sensospot fluorescence image (Figure 17).

[0068] Amplification and detection of Serratia marcescens at a single target region was demonstrated, with a significant delta ISFET voltage signal detected that was greater than that of the control anchored primers.

[0069] Example 2: Multiple Localized Anchored Primer Amplification and Detection (LAPAD) Target: Schizosaccharomyces pombe Chip ID: NM-248-0172 The anchored primer sequences for Example 2 are shown in Table 5. [Table 5]

[0070] The average delta signal from the ISFET of Example 2 is shown in FIG.

[0071] The delta signals from all ISFETs for anchored primer targets 1-4 positive are shown in FIG.

[0072] The delta signals from all ISFETs for the anchored primer negative are shown in FIG.

[0073] The delta signals from all ISFETs for the anchored primer control are shown in FIG.

[0074] The results of the average ISFET signals for the positive, negative and control primers are shown in Figure 22. ISFET signals ranging from 1.5 to 4.4 dmV were detected for the four positive anchored primers, which is much lower than the previous signal intensity and the average ISFET signal for the control anchored primer, which was 34.6 dmV.

[0075] As seen in the gel electrophoresis image, solution-phase amplicons were generated after PCR for Schizosaccharomyces pombe (Figure 23). The generated on-chip amplicons are not visible for the positive anchored primer in the Sensospot fluorescence image (Figure 24). However, the control anchored primer is visible.

[0076] Amplification and detection of Schizosaccharomyces pombe at multiple target regions was demonstrated. The present invention includes the following preferred embodiments. (1) A method for the amplification and analysis of nucleic acid sequences, comprising: a. taking a system having a liquid volume in contact with a solid support, the liquid volume comprising a nucleic acid sample, two or more non-immobilized amplification primers, and reagents for nucleic acid amplification, the solid support having one or more immobilized amplification primers; b. performing a nucleic acid amplification reaction using a non-immobilized amplification primer to generate a first non-immobilized nucleic acid amplification product; c. further amplifying the first non-immobilized nucleic acid amplification product using one or more immobilized amplification primers to produce an immobilized second nucleic acid amplification product; di examining in real time the localized signal generated at the location of the immobilized second nucleic acid amplification product; or ii. removing the liquid volume and the first non-immobilized amplification product and replacing it with a new liquid volume having a primer that hybridizes to the immobilized second nucleic acid amplification product; Either of the following, and 1. Direct detection of hybridized primers, or 2. Flowing a solution containing at least one nucleotide over the immobilized second amplification product to extend the hybridized primer. Any of the following, and e. Detecting the presence, absence, or sequence of a second nucleic acid amplification product. The above method. (2) The method according to (1), wherein the non-immobilized amplification primers in the liquid volume are released from the solid phase prior to amplification. (3) The method according to claim 1 or 2, wherein the nucleic acid amplification reaction using non-immobilized primers uses two or more pairs of non-immobilized primers. (4) The method according to (2), wherein the amplification is carried out by thermal cycling. (5) The method according to any one of (1) to (3), wherein the nucleic acid amplification reaction using a non-immobilized primer is an isothermal amplification. (6) The method according to (5), wherein the second amplification product is generated by further isothermal amplification. (7) The method according to (5), wherein the second amplification product is generated by thermal cycling. (8) The method according to any one of (1) to (7), wherein the amplification of the immobilized second nucleic acid amplification product is carried out by examining a localization signal generated in real time. (9) The method according to (8), wherein the two primers in the solution are present at different concentrations to effect asymmetric amplification. (10) The method according to any one of (1) to (7), wherein the primer hybridized to the second nucleic acid amplification product is fluorescently labeled. (11) The method according to any one of (1) to (7), wherein the detection of the amplification product involves detection of protons released upon incorporation of a nucleotide. (12) The method according to (11), wherein the detection uses an ISFET sensor. (13) The method according to any one of (1) to (7), wherein the detection of the amplification product comprises a fluorescent reporter probe and subsequent measurement of fluorescence. (14) The method according to any one of (1) to (13), wherein the first and second amplification products are obtained using amplification primers having the same nucleic acid sequence. (15) The method according to any one of (1) to (13), wherein the immobilized amplification primer amplifies an internal portion of the first amplification product such that the second amplification product is shorter than the first amplification product. (16) The method of any of (1) to (15), wherein the first liquid volume is exposed to two or more immobilized amplification primers, each primer amplifying a different sequence. (17) The method of (16), wherein two or more immobilized amplification primers differ by a single base, thereby detecting a single base variant in the first nucleic acid amplification product. (18) The method according to (16) or (17), wherein the immobilized primers are on an open microarray. (19) The method according to (16) or (17), wherein the immobilized primers are in separate wells. (20) A system for amplification and detection of nucleic acid sequences comprising a liquid volume in contact with a solid support, the liquid volume comprising a nucleic acid sample, one or more non-immobilized amplification primers, and reagents for nucleic acid amplification, and the solid support carrying one or more immobilized amplification primers, the system comprising a sensor for detecting the amplification product. (21) The system described in (20), wherein the sensor is an optical sensor. (22) The system described in (20), wherein the sensor is an electrical sensor. (23) The system according to (20)0 or (22), wherein the sensor is a CMOS sensor. (24) The system described in (23), comprising a plurality of ISFET sensors and two or more types of immobilized primers, each ISFET sensor comprising a single immobilized primer. [Sequence List Free Text]

[0077] Sequence Listing 3 <223> The C at position 1 is attached to spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer, which is attached to a 6-carbon amino modifier (5AmMC6). Sequence Listing 4 <223> The T at position 1 is attached to spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer, which is attached to a 6-carbon amino modifier (5AmMC6). Sequence Listing 5 <223> The G at position 1 is attached to spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer, which is attached to a 6-carbon amino modifier (5AmMC6). Sequence Listing 6 <223> The G at position 1 is attached to spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer, which is attached to a 6-carbon amino modifier (5AmMC6). Sequence Listing 7 <223> The T at position 1 is attached to a six carbon amino modifier (5AmMC6). Sequence Listing 8 <223> The T at position 1 is conjugated to a fluorescent dye (5Cy3). Sequence Listing 11 <223> Between the C at position 20 and the A at position 21 is spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer. Sequence Listing 12 <223> Between C at position 20 and G at position 21 is spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer. Sequence Listing 13 <223> Between the 20th and 21st C is an 18-atom hexaethylene glycol spacer, spacer 18 (iSp18). Sequence Listing 14 <223> Between C at position 20 and G at position 21 is spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer. Sequence Listing 15 <223> Between the C at position 20 and the A at position 21 is spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer. Sequence Listing 16 <223> Between C at position 20 and G at position 21 is spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer. Sequence Listing 17 <223> Between the C at position 20 and the T at position 21 is spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer. Sequence Listing 18 <223> Between the C at position 20 and the A at position 21 is spacer 18 (iSp18), an 18-atom hexaethylene glycol spacer. Sequence Listing 20 <223> The C at position 1 is bound to a fluorescent dye (5Cy3).

Claims

1. 1. A method for the amplification and analysis of a nucleic acid sequence, comprising: a. taking a system having a liquid volume in contact with a solid support, the liquid volume comprising a nucleic acid sample, two or more non-immobilized amplification primers, and reagents for nucleic acid amplification, the solid support having one or more immobilized amplification primers; b. performing a nucleic acid amplification reaction using a non-immobilized amplification primer to generate a first non-immobilized nucleic acid amplification product; c. further amplifying the first non-immobilized nucleic acid amplification product using one or more immobilized amplification primers to generate an immobilized second nucleic acid amplification product; d. i. examining in real time the localized signal generated at the location of the immobilized second nucleic acid amplification product; or ii. removing the liquid volume and the first non-immobilized amplification product and replacing it with a new liquid volume having a primer that hybridizes to the immobilized second nucleic acid amplification product; Either of the following, and 1. Direct detection of hybridized primers, or 2. Flowing a solution containing at least one nucleotide over the immobilized second amplification product to extend the hybridized primer. Any of the following, and e. Detecting the presence, absence, or sequence of a second nucleic acid amplification product. The above method.

2. The method of claim 1 , wherein the non-immobilized amplification primers in the liquid volume are released from the solid phase prior to amplification.

3. The method according to claim 1 or 2, wherein the nucleic acid amplification reaction using non-immobilized primers uses two or more pairs of non-immobilized primers.

4. The method of claim 1 , wherein the amplification is performed by thermal cycling.

5. The method according to any one of claims 1 to 3, wherein the nucleic acid amplification reaction using a non-immobilized primer is an isothermal amplification.

6. The method of claim 5 , wherein the second amplification product is generated by a further isothermal amplification.

7. The method of claim 5 , wherein the second amplification product is generated by thermal cycling.

8. The method according to any one of claims 1 to 7, wherein the amplification of the immobilised second nucleic acid amplification product is carried out by examining a localised signal generated in real time.

9. 9. The method of claim 8, wherein the two primers in solution are present at different concentrations to provide asymmetric amplification.

10. The method according to any one of claims 1 to 7, wherein the primer hybridized to the second nucleic acid amplification product is fluorescently labeled.

11. The method according to any one of claims 1 to 9, wherein the detection of the amplification product uses detection of protons released upon nucleotide incorporation.

12. The method of claim 11 , wherein the detection uses an ISFET sensor.

13. The method of any one of claims 1 to 7, wherein detection of the amplification product comprises a fluorescent reporter probe and subsequent measurement of fluorescence.

14. The method according to any one of claims 1 to 13, wherein the first and second amplification products are obtained using amplification primers having the same nucleic acid sequence.

15. The method of any one of claims 1 to 13, wherein the immobilized amplification primer amplifies an internal portion of the first amplification product such that the second amplification product is shorter than the first amplification product.

16. 16. The method of any one of claims 1 to 15, wherein the first liquid volume is exposed to two or more immobilized amplification primers, each primer amplifying a different sequence.

17. 17. The method of claim 16, wherein two or more immobilized amplification primers differ by a single base, thereby detecting a single base variant in the first nucleic acid amplification product.

18. 18. The method of claim 16 or claim 17, wherein the immobilized primers are on an open microarray.

19. 18. The method of claim 16 or claim 17, wherein the immobilized primers are in separate wells.

20. A system for amplification and detection of nucleic acid sequences comprising a liquid volume in contact with a solid support, the liquid volume containing a nucleic acid sample, one or more non-immobilized amplification primers and reagents for nucleic acid amplification, and the solid support carrying one or more immobilized amplification primers, the system comprising a sensor for detecting the amplification products.

21. The system of claim 20 , wherein the sensor is an optical sensor.

22. 21. The system of claim 20, wherein the sensor is an electrical sensor.

23. 23. A system according to claim 20 or claim 22, wherein the sensor is a CMOS sensor.

24. 24. The system of claim 23, comprising a plurality of ISFET sensors and two or more immobilized primers, each ISFET sensor comprising a single immobilized primer.

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