Combined liquid and solid phase DNA amplification

Abstract

The present application relates to combined liquid and solid phase DNA amplification, and specifically describes a method for efficient amplification and detection of certain nucleic acid sequences in a population. The selected population can be further characterized, for example, by sequencing. The method includes combined liquid and solid phase amplification.

Description

[0001] This patent application is a divisional application of the patent application with application number 2020800342267, application date May 7, 2020, and invention title "Combined Liquid and Solid Phase DNA Amplification". Technical Field

[0002] This invention relates to methods for sequence-specific amplification of subpopulations of nucleic acid fragments from a broader population of nucleic acid sequences. The invention specifically detects only the desired target sequence. Aspects of the invention relate to nucleic acid constructs for such methods. The invention describes methods for efficiently targeting and amplifying specific nucleic acid sequences within a population. Background Technology

[0003] Identifying pathogens in blood and other biological samples is crucial for the effective treatment of a variety of disease states, including sepsis. In the UK, five people die from sepsis every hour, and 25% of all sepsis survivors suffer permanent, life-altering consequences. Early detection and identification of the pathogens causing bacterial infections are essential for preventing sepsis outbreaks and for successful treatment. Existing methods for detecting and identifying bloodborne infections include blood cultures and antibiotic susceptibility testing. These methods involve cell cultures, which are expensive in both time and money. Often, septic shock occurs before cell culture results are available.

[0004] The application of existing molecular detection methods, such as PCR, which identify pathogens in samples by analyzing their nucleic acids, is limited by low overall sensitivity levels. This is particularly problematic when analyzing human samples due to the presence of a large number of non-target (human) nucleic acids. These non-target nucleic acids can inhibit downstream purification and amplification of target (pathogen) nucleic acids, leading to false negative results or false positive readings of pathogen nucleic acids due to non-specific amplification.

[0005] For example, several antibiotic resistance genes exist, which have multiple variants due to point mutations or other polymorphisms, and the presence of a specific mutation can lead to different treatment strategies compared to another mutation. Therefore, it is important to identify these polymorphisms in a timely and cost-effective manner. Existing identification strategies using PCR and other amplification reactions have limited capabilities, and while laboratories can employ sequencing to elucidate internal sequences and identify polymorphisms—this is time-consuming and expensive. Therefore, selectively amplifying populations of target nucleic acids relative to non-target nucleic acids is of significant value to the pharmaceutical industry and research.

[0006] Amplification reactions typically rely on a pair of primers for each desired sequence to be amplified. While several pairs of non-immobilized primers can be combined, combining a large number of primer pairs is usually impractical due to cross-hybridization between primers. Therefore, multiplex PCR is typically performed using individual primers in separate solution fractions, requiring a large number of samples. This invention describes an improvement to the amplification reaction that enables sequence-selective amplification of a variety of different primers in a single sample volume.

[0007] A method for emulsion PCR is also disclosed, in which a single template is diluted such that each bubble in the oil emulsion contains, on average, less than one template molecule. Water droplets may include a single immobilized primer, with a second primer in solution. Thus, amplification is performed using a mixture of two primers, one immobilized and the other in solution. However, unlike the method described herein which further amplifies the first amplification product, this method produces only a single amplification product in each reaction fraction.

[0008] It is also known that two of these primers are used in immobilized amplification methods, such as Illumina cluster-based bridging amplification. In this case, no primers are present in the free solution. Summary of the Invention

[0009] This invention provides a biphasic amplification reaction comprising both solid-phase and liquid-phase (thermal cycling or isothermal) amplification reactions. This amplification reaction can utilize combinations of immobilized and non-immobilized primers and target DNA templates to simultaneously generate DNA amplicones in solution and on a surface. The presence or absence of amplicones can be detected in a post-amplification endpoint assay or in real time. This biphasic approach differs from existing techniques, including emulsion PCR, because it incorporates both solid-phase (i.e., surface-based) amplification and liquid-phase amplification.

[0010] Nucleic acid samples can be amplified using one or more immobilized primers. The DNA generated in this liquid phase, as well as the target DNA template, can also be used as, or subsequently used as, a template for additional reactions on 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 with different sequences at known locations and using a detection system capable of identifying those locations (e.g., using ISFETs on a CMOS IC chip or an array based on a fluorescence / optical imaging system), the readings provide further discrimination capability to the system.

[0011] Here, we propose a novel method to achieve multiplex sequence-specific amplification of subpopulations of nucleic acid fragments from a broader population of nucleic acid sequences.

[0012] This application describes a method for amplifying and analyzing nucleic acid sequences, the method comprising:

[0013] a. Contact a system having a liquid volume with a solid carrier, wherein the liquid volume contains a nucleic acid sample, two or more immobilized amplification primers and reagents for nucleic acid amplification, and the solid carrier has one or more immobilized amplification primers;

[0014] b. Use non-immobilized amplification primers to perform a nucleic acid amplification reaction to produce a first non-immobilized nucleic acid amplification product;

[0015] c. Further amplify the first non-immobilized nucleic acid amplification product using one or more immobilized amplification primers to generate an immobilized second nucleic acid amplification product;

[0016] d. One of the following two

[0017] i. Real-time query of the localization signal generated at the immobilized second nucleic acid amplification product, or

[0018] ii. Remove the liquid portion and the first non-immobilized amplification product, and replace it with a new liquid portion having primers that hybridize with the immobilized second nucleic acid amplification product, and one of the following.

[0019] 1. Direct detection of hybridization primers, or

[0020] 2. Pass a solution containing at least one nucleotide through an immobilized second amplification product to extend the hybridization primers, and

[0021] e. Detect the presence, absence, or sequence of the second nucleic acid amplification product.

[0022] This method can be performed as follows, wherein, prior to amplification, the immobilized amplification primers are released from the liquid portion of the solid phase. Nucleic acid amplification reactions using immobilized primers may employ two or more pairs of immobilized primers. Nucleic acid amplification at any stage can be isothermal, or it can be performed through thermal cycling.

[0023] According to this method, the primers hybridizing with the second nucleic acid amplification product can be fluorescently labeled, and subsequent fluorescence assays can be performed. Alternatively, the method can be carried out in which the amplification product is detected by, for example, an ISFET sensor by detecting protons released upon nucleotide incorporation.

[0024] This method can be performed in such a way that amplification primers with the same nucleic acid sequence are used to obtain a first amplification product and a second amplification product.

[0025] The method may also include immobilized amplification primers amplifying the internal segments of the first amplification product, such that the second amplification product is shorter than the first amplification product.

[0026] The method may also include exposing the same first liquid fraction to two or more immobilized amplification primers, each primer amplifying a different sequence. The two or more immobilized amplification primers may differ by a single base, thereby detecting single-base variants during the amplification of the first nucleic acid.

[0027] The immobilized primers in this method can be located on an open microarray or in individual wells. Individual wells can be simultaneously exposed to the same reagent motif. If the wells contain immobilized primers with different sequences, different amplicones can be generated in different wells, thus enabling multiplex amplification within a single liquid motif.

[0028] Another aspect of the present invention provides a system for amplifying and detecting nucleic acid sequences, comprising a liquid portion in contact with a solid carrier, wherein the liquid portion contains a nucleic acid sample, two or more immobilized amplification primers and reagents for nucleic acid amplification, and the solid carrier has one or more immobilized amplification primers, wherein the system includes a sensor for detecting amplification products.

[0029] The amplification products can be detected using optical or electrical methods. For optical detection, the amplification products need to be labeled with fluorescent markers, and then the markers can be detected using optical methods.

[0030] Whether optical or electrical output is detected, it can be sensed via an array of silicon-based sensors, such as field-effect transistors (FETs). The chosen FET can be any suitable CMOS chip, including (but not limited to) ion-sensitive field-effect transistors (ISFETs). The sensor is configured to integrate signals detected in one or both time and space to determine the presence or absence of amplification products, or in some cases, the amount of amplification products present. Unlike some deployments of optical arrays, the sensor is not configured to output an image. Attached Figure Description

[0031] Figure 1 This diagram illustrates a single-target anchored primer amplification and detection (LAPAD).

[0032] Figure 2 This diagram illustrates multiple localization anchored primer amplification and detection (LAPAD).

[0033] Figure 3The diagram illustrates the elution process. This occurs in an open system requiring on-chip flow capability. During the amplification reaction, product amplicones in the liquid phase fall onto solid-phase primers, which extend to generate numerous extended amplicons on the chip surface. After amplification, the amplification products and reagents are washed away, leaving single-stranded amplicons on the chip surface. The eluted primers and enzyme are loaded onto the chip, and after a short hybridization step, all four nucleotides flow in together, generating a proton burst on those ISFETs with extended oligonucleotides. This proton burst is clearly observed in the most simplified data processing.

[0034] Figure 4 The elution workflow and the resulting data are illustrated. The first row represents the amplification phase of the experiment, generating a cluster of extended amplicons on the chip surface. This is followed, for example, by a heated dehybridization step to generate single-stranded amplicons on the chip surface, with no product in the liquid phase. After elution of primers and enzyme hybridization, all four nucleotides flow through the chip, generating signals on those ISFETs that have been specifically extended.

[0035] Figure 5 The sample ISFET data are shown, illustrating the elution signals generated from the three target templates after PCR. The data presented represent Escherichia coli (80 bases of product, therefore a maximum of 60 bases eluted), Enterococcus faecalis, and Serratia marcescens after target-specific amplification. Unmodified data, showing all ISFETs.

[0036] Figure 6 A multiplex microarray is shown with three anchor primers and a positive template control. PCR was performed with the Ef template, followed by elution. The heatmap shows the ISFET voltage measured ~15 seconds after the nucleotide flow. The ISFETs can be seen to match the spotting pattern and correspond to both the Ef and positive controls. The traces on the right show examples of the ISFET responses for each ISFET group.

[0037] Figure 7The sequencing implementation scheme is illustrated. Instead of adding all four nucleotides for elution, they are added sequentially. This occurs in an open system requiring flow capability on the chip. During the amplification reaction, product amplicones in the liquid phase fall onto solid-phase primers, which extend to generate numerous extended amplicons on the chip surface. After amplification, the amplification products and reagents are washed away to leave 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 flow in individually, generating burst pulses of protons on those ISFETs containing the amplification products and the correct template bases. Single-base resolution can be achieved.

[0038] Figure 8 The sequencing workflow and the resulting data are illustrated. The first row represents the amplification phase of the experiment, where a cluster of extended amplicons is generated on the chip surface. This is followed by, for example, a heated dehybridization step to generate single-stranded amplicons on the chip surface, with no product in the liquid phase. After eluting primers and enzyme hybridization, all four nucleotides flow through the chip individually, generating signals on those ISFETs incorporating the nucleotides, corresponding to the generation of amplified products.

[0039] Figure 9 A real-time detection workflow is illustrated. It can occur on platforms with no or minimal fluid flow after chip amplification. Reactions can be performed in one or more permanently sealed reaction chambers. During the amplification reaction, product amplicons in the liquid phase fall onto a solid-phase oligomer, modifying and extending the specific oligomer. In a sealed system with low buffering, protons will be generated in the near-solid phase during the extension phase of each cycle of the amplification reaction. Pushing for asymmetry to generate a significant excess of forward primers in the liquid phase will effectively generate “micro-elution” bursts of protons in each cycle, which should be detectable in subsequent cycles. As an alternative to primer asymmetry at the start of the amplification reaction, additional primers can be released or added during the amplification reaction or at set time points. The amplification reaction will be performed in a low buffer solution to enable real-time detection of the pH signal.

[0040] Figure 10 Real-time detection data are shown. The chip was batch-sampled with multiple anchored primers, and PCR (3:1 asymmetric) with a 6-compartment gasket (3 fluid-sealed compartments) was eluted in a flow stage with a single compartment. Liquid chromatography shows real-time pH signals, indicating the reaction was in progress. As confirmation of post-PCR amplification, fluorescent tags targeting the distal ends of the amplicon were used as probes on the chip, demonstrating that the anchored primers extended during the PCR reaction.

[0041] Figure 11 The average δ signal for the ISFET in Example 1 is shown.

[0042] Figure 12 The δ signal of all ISFETs for the anchored primer target positive of Example 1 is shown.

[0043] Figure 13 The δ signal of all ISFETs for the anchored primer target negative of Example 1 is shown.

[0044] Figure 14 The δ signal of the entire ISFET for the anchored primer control of Example 1 is shown.

[0045] Figure 15 The average ISFET signal results for the positive, negative, and control primers of Example 1 are shown.

[0046] Figure 16 The image shows a gel electrophoresis diagram of liquid phase amplicon generation after PCR for Serratia marcescens of Example 1.

[0047] Figure 17 A Sensospot fluorescence image for Example 1 is shown.

[0048] Figure 18 The average δ signal for the ISFET in Example 2 is shown.

[0049] Figure 19 The δ signals for anchor primer targets 1 to 4 of Example 2 are shown.

[0050] Figure 20 The δ signal for the entire ISFET is shown for the anchor primer negative of Example 2.

[0051] Figure 21 The δ signal of the entire ISFET for the anchored primer control of Example 2 is shown.

[0052] Figure 22 The average ISFET signal results for the positive, negative, and control samples of Example 2 are shown.

[0053] Figure 23 The image shown is a gel electrophoresis image for Example 2.

[0054] Figure 24 The image shown is a Sensospot fluorescence image for Example 2. Detailed Implementation

[0055] This application describes a system capable of generating readings of different sequences from the same reaction motif.

[0056] In short, the system comprises a biphasic amplification reaction in which a liquid-phase (PCR or isothermal) amplification reaction occurs in the presence of primers immobilized on a solid phase that further participate in generating amplification products. The liquid-phase (PCR or isothermal) amplification reaction can use one or more primers and a target nucleic acid template to generate nucleic acid amplification products in solution, and the system detects the corresponding signal when sufficient products have been generated. This liquid-phase generated nucleic acid amplification product, along with the target nucleic acid template, can also serve as, or subsequently as, a template for additional reactions carried out on the solid phase, whereby, as part of the amplification reaction, one or more immobilized primers interact with the liquid-phase template to generate a nucleic acid sequence that can subsequently be detected by the system. By immobilizing the primers at known locations and using a detection system capable of identifying those locations (e.g., using an ISFET on a CMOS IC chip or an array-type optical imaging system), the resulting readings enable the selective amplification and detection of many amplicones in the same device.

[0057] The amplification reaction is carried out in the liquid phase in the presence of one or more other primers immobilized on a solid phase. The immobilized primers can be sequences identical to one or more liquid phase primers, or they can be internal sequences corresponding to the nucleic acid template generated in the liquid phase. Sequences identical to one or more liquid phase primers can be called a second time using the same reaction components, while a second call to the internal sequence corresponding to the nucleic acid template generated in the liquid phase increases confirmation and improves confidence, i.e., the correct amplification product has been generated and / or further provides information about the internal sequence of the target template.

[0058] The detection modality described in this article can utilize ISFET CMOS technology, but is also applicable to existing nucleic acid amplification reactions and microarray methods, as well as custom systems integrating real-time detection of both liquid-phase and solid-phase reactions. The reproducibility potential of ISFET CMOS allows for numerous repetitions of each assay and multiple target genes for each target, thereby increasing system confidence through multiple target-dependent readings. This design also facilitates the inclusion of positive and negative controls in each reaction vessel, ensuring correct adjudication when controls are required.

[0059] The following are different implementation schemes for combining liquid-phase and solid-phase amplification reactions.

[0060] DNA amplification readings

[0061] The immobilized primers can be the same as or similar to one or more liquid-phase primers and participate in the amplification reaction, resulting in an increased signal generated by the immobilized primers compared to amplification without liquid-phase primers, thereby increasing the confidence of the result, but without adding further information to the generated sequence. For example, asymmetric PCR in the liquid phase can be compensated by having the primers consumed in the liquid phase remain on the solid phase. As the amplification reaction proceeds, the generated liquid-phase products interact with the solid phase, thereby generating both liquid-phase and solid-phase amplicones. The immobilized amplicon can be detected. The presence of such readouts at multiple sites on the immobilized primers increases the confidence that the subsequently amplified material is definitively present in the sample. If immobilized primers with different sequences fail to produce a positive result, the confidence of the result is increased.

[0062] With internally confirmed DNA amplification

[0063] The immobilized primers can correspond to the internal sequences of the DNA template generated in the liquid phase, so that any signal generated on the solid phase is a positive confirmation that the correct product has been generated in the liquid phase. This confirmation proves that the liquid phase amplicon is not generated by substances such as primer dimers, mismatched primers, or other non-specific reactions (e.g., signals generated by interaction with host (human) DNA when the target is a pathogen in the host sample), and provides a significant benefit over standard confirmatory tests such as melting curve analysis and electrophoretic gel imaging, because additional sequence information is inferred from the readings.

[0064] Internal validation signals are similar to standard laboratory validation procedures, such as melting curve analysis and electrophoretic gel imaging.

[0065] Using immobilized primers corresponding to the internal sequence in the target template gives the system further specificity. Thus, the combination of liquid-phase and solid-phase reactions identifies more sequence information than liquid-phase reactions alone, thereby improving the confidence of the results compared to liquid-phase reactions alone.

[0066] DNA amplification with internal sequence information

[0067] In addition to the already identified confirmation steps, there is the potential to allow for multiple immobilized primers corresponding to different regions of the liquid-phase amplification product, such that one or more immobilized primers can be used to provide internal information beyond simply confirming that the correct product has been generated in the liquid phase.

[0068] This can take the simple form of multiple internal primers derived from the amplification product, each internal primer increasing the confidence of the inferred result by providing more than one sequence alignment. Alternatively, in more advanced versions, it can be used to identify internal variants within a target gene corresponding to point mutations and other polymorphisms. For example, several antibiotic resistance genes exist, which have multiple variants due to point mutations or other polymorphisms, and the presence of a particular mutation can lead to a different treatment strategy compared to another mutation. Therefore, it is important to identify these polymorphisms in a timely and cost-effective manner. Existing identification strategies using PCR and other amplification reactions have limited capabilities, and while sequencing can be used in laboratories to elucidate internal sequences and identify polymorphisms, this is time-consuming, expensive, and often not feasible.

[0069] In this implementation, single primer pairs can be used in the liquid phase to amplify all variants of a gene, while one or more immobilized primers corresponding to point mutations or polymorphisms can be used on the solid phase to identify one or more variants. The combination of internally immobilized primers provides users with rich information about the overall sequence of the amplified gene and enables easier and faster clinical decision-making.

[0070] Washing (including multiple washes)

[0071] Amplification reactions are typically performed in high ionic strength buffers. For sequencing systems that determine the proton release of incorporated nucleotides, the amplification products can be detected if a buffer with low buffering capacity is used instead of the amplification buffer. Alternatively, the amplification products can be detected using fluorescently labeled primers or via fluorescently labeled nucleotides.

[0072] Elution occurs in open systems requiring flow capability on the chip. During the amplification reaction, product amplicons in the liquid phase fall onto solid-phase primers, which extend to generate numerous extended amplicons on the chip surface. After amplification, the amplification products and reagents are washed away to leave single-stranded amplicons on the chip surface. The eluted primers and enzyme are loaded onto the chip, and after a short hybridization step, all four nucleotides flow in together, generating a burst of protons on those ISFETs with extended oligonucleotides. This burst of protons is clearly observed in the most simplified data processing.

[0073] For electron / proton detection, at the end of the aforementioned amplification phase involving both liquid and solid-phase reactions, the liquid phase component is flushed out of the column and replaced with a solution having low buffering capacity. Then, primers and enzymes, or combinations of primers and enzymes, are followed by a nucleotide stream, which releases protons upon incorporation of nucleotides and generates a corresponding signal on the detection platform, such as a mV change on a correlated ISFET. Primers used for elution can correspond to any position within the amplification template, including universal sequences added during early amplification reactions. Using amplicon-specific primers in solution provides increased confidence in the results by confirming that the correct material has been generated on the solid phase, but may increase system complexity due to higher multiplicity. Incorporating universal sequences in early amplification reactions may limit the scale of liquid-phase multiplex amplification, thereby improving reaction efficiency. However, this may limit read confidence due to the potential for misfiring of universal sequences.

[0074] By knowing the location of a specific immobilized primer, the identification of a target can be determined when the protons generated during the elution reaction produce a signal on a specific ISFET sensor immobilized with that specific primer. In this embodiment, it is possible to repeat each experimental target multiple times, where the scale of multiplicity is limited by spatial separation, the number of pores and sensors per device, and bioinformatics challenges.

[0075] This system can utilize a single or multiple elution reaction, with or without a dehybridization step between each run. Additional elutions can be used to query earlier signals, increasing the confidence of the internal sequence. For example, if a universal sequence is used in the early amplification reaction, the first elution can use the universal sequence to identify which assays have generated signals, followed by elution with specific primers corresponding to those specific assays to confirm the correct product was produced.

[0076] Since primers with different sequences can be fixed at different positions on a solid vector, multiple target sequences can be analyzed in parallel.

[0077] Sequence information (one or more bases at a time)

[0078] At the end of the amplification phase described above, the liquid phase components can be flushed out of the column and replaced with a low-buffered solution, as in the elution workflow. Then, sequentially, primers and enzymes, or combinations of primers and enzymes, followed by streams of single nucleotides, or streams of two or more nucleotides, releasing protons as the correct complementary nucleotides flow through the chip and are incorporated. This workflow provides the richest information across the entire implementation because it provides sequence information, rather than relying solely on the alignment of primers with their corresponding templates. This workflow not only confirms that correct alignment has occurred and that the correct product has been generated on the solid phase, but also indicates the internal sequence by inference or direct identification. Similar methods can be used, as discussed in the elution implementation, where multiple runs can provide further information, and workflows combining elution with this method using one or more nucleotides.

[0079] Real-time detection

[0080] The amplification process can be detected in real time by measuring the extension of immobilized primers. The reaction can be carried out in one or more permanently sealed reaction chambers. During the amplification reaction, product amplicones in the liquid phase fall onto solid-phase oligomers, modifying and extending specific oligomers. In a sealed system with low buffering, protons are generated near the solid phase during the extension phase of each cycle of the amplification reaction, and these protons can be detected.

[0081] Amplification can be asymmetric, where two primers are present in solution at different concentrations, resulting in predominantly single-stranded amplification products due to the lack of complementary primers for their extension. Driving the asymmetry to generate a significant excess of the forward primer in the liquid phase will effectively generate proton “micro-elution” bursts in each cycle, which should be detectable in subsequent cycles. As an alternative to primer asymmetry at the start of the amplification reaction, additional primers can be released or added during the amplification reaction or at set time points. The amplification reaction will be carried out in a low buffer solution to allow for real-time monitoring of the pH signal.

[0082] The source nucleic acid may be a genomic polynucleotide. The source material may be eukaryotic, prokaryotic, or archaea. One or more source materials may be provided. The source nucleic acid may represent a segment of the genome; for example, a single chromosome or a single genomic locus (e.g., for rapid sequencing of allelic polymorphisms). In certain embodiments, the amplification of pathogenic material in the sample is specific. For example, bacterial or viral nucleic acids present in a human sample may be selected for amplification. The template may be DNA, RNA, or a copy of its cDNA.

[0083] Biological material can be amplified in solution prior to the claimed amplification reaction. Therefore, the material may have already undergone sequence-based amplification steps prior to this invention. Alternatively, the sample can be processed to ligate a common sequence shared by one or both ends of the entire strand of the sample. This common sequence can be used as a universal sequence for hybridization with shared primers that may not be naturally occurring.

[0084] The invention described herein allows for four or more levels of amplification specificity. The first amplification can be performed in solution. The second amplification can be performed using non-immobilized primers. The third amplification can be performed using immobilized primers with sequences different from the second amplification primers. The presence of the amplicon can be detected using primer specificity against the amplicon generated by the immobilized primers. Therefore, four levels of amplification specificity can be used, ensuring that the final amplicon is detected only when the starting sequence is absolutely and definitively present in the sample. Thus, false positives from detections of closely related but undesirable sequences can be eliminated.

[0085] Immobilizing nucleic acids onto surfaces is routine. Any method of immobilization, whether covalent or non-covalent, can be used. Ideally, the immobilization is stable after multiple cycles of thermal cycling to temperatures that cause nucleic acid chain separation. Specific methods of immobilization include UV-induced crosslinking or immobilization via the formation of amide or thiophosphate bonds.

[0086] Example

[0087] Experimental data have been collected for single-target anchoring primer amplification and detection (LAPAD) of Serratia marcescens (supplier: NCTC, catalog number 27137) in a single target region and for multiple-target anchoring primer amplification and detection (LAPAD) of Schizostomia spp. (supplier: ATCC, catalog number 26189) in multiple target regions.

[0088] The diagrams for single LAPAD and multiple LAPAD are as follows: Figure 1 and Figure 2 As shown.

[0089] Example 1: Single-target anchored primer amplification and detection (LAPAD)

[0090] Serratia marcescens was amplified and detected in a single target region. The target region located at the 3' end of the amplified template DNA hybridized with the complementary anchoring primer target. During amplification, this anchoring primer target was extended, and the same target region was detected in the next stage during extension. The primer / oligonucleotide sequences used are shown in Table 1.

[0091] Table 1: Oligonucleotide sequences

[0092]

[0093] Example 2: Multiplexed Targeted Amplification and Detection (LAPAD)

[0094] Amplification and detection of *Schizosaccharomyces cerevisiae* in multiple target regions.

[0095] Target region 1, a few nucleotides after the 3' end of the template DNA, is hybridized to complementary anchor primer target 1. Target region 2, a few bases after target region 1 and pointing towards the 5' end of the template DNA, is hybridized to complementary anchor primer target 2. Target region 3, a few bases after target region 2 and pointing towards the 5' end of the template DNA, is hybridized to complementary anchor primer target 3. Target region 4, a few bases after target region 3 and pointing towards the 5' end of the template DNA, is hybridized to complementary anchor primer target 4. During amplification, these four anchor primer targets with hybridized DNA templates are extended, and then in the next stage, the same target regions are detected during extension. The primer / oligonucleotide sequences used are shown in Table 1.

[0096] Experimental methods

[0097] A chip platform consisting of a '004' Ta2O5 CMOS chip composed of ISFETs and enclosed in an SU-8 well is anchored with primers using UV-based crosslinking surface chemistry. A manifold is mounted on the chip surface to include fluids for reactions to occur on the chip surface.

[0098] PCR was performed to generate DNA for detection. The PCR reagent formulation, with a final volume of 50 μl, is shown in Table 2. The thermal cycling conditions used are shown in Table 3.

[0099] Table 2: PCR Reagent Formulations

[0100]

[0101] Table 3: Thermal Cycling Conditions

[0102]

[0103] After PCR, the chip was washed with washing buffer (0.06x saline-sodium citrate, 0.06% Tween 20), then the DNA was chemically dehybridized with 20mM NaOH for 5 minutes, and then washed again with washing buffer.

[0104] The eluted primer mixture (sequences shown in Table 1) was then added to the chip surface. 3.33 μM of fluorescently labeled (Cy3) primers were added to annealing buffer (5 mM magnesium acetate, 150 mM sodium chloride, 20 mM Tris pH 7.5, 0.01% Tween 20).

[0105] The chip containing the elution primer mixture was then heated at 95°C for 2 minutes, followed by heating at 58°C for 5 minutes, and incubated at room temperature for 15 minutes. Then, it was loaded with enzyme loading buffer (1x... 0.06% Tween 20) washing chip.

[0106] The enzyme mixture was then added to the surface of the chip by adding 25 units / μL of a custom Bst large fragment DNA polymerase to the enzyme loading buffer. The chip was then incubated at room temperature for 5 minutes.

[0107] Deoxyribonucleotide triphosphates (dNTPs) were incorporated using a custom flow system that allows dNTPs to flow onto the chip surface. 12.5 μM of each of deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate were added to a non-carbonated RMD buffer (10 mM magnesium chloride, 25 mM sodium chloride, 0.025% Tergitol). The dNTP mixture was adjusted to pH 8 and maintained by keeping the fluid under nitrogen atmosphere.

[0108] ISFET sensor detection

[0109] The original ISFET sensor voltage output data and anchor primer spotting Figure 1 The data was processed using a custom data analysis pipeline (Galaxy Elution Pipeline v1.3.15). The voltage signal peak height of the ISFET was detected, and the voltage signal peak height of the anchor primer ISFET was subtracted from the voltage signal peak height of the adjacent upstream blank ISFET. This was done to remove system noise from the fluid flow, including noise that could mask the detection results. The voltage output of this δISFET sensor from all specific anchor primer sensors was averaged to determine the signal detection result.

[0110] E-gel electrophoresis analysis

[0111] The prepared E-gels (48-well 2% agarose gels stained with ethidium bromide) were used for qualitative analysis by liquid-phase PCR. Samples were collected from the chip surface after PCR, and 1 μL of sample was added to 1x E-gel loading dye before loading onto the E-gel wells. Gradient loading was also performed to compare amplicon sizes. The gels were run on an E-gel substrate with an electric field for 10 minutes to allow DNA amplicon migration according to their size.

[0112] Sensospot fluorescence imaging

[0113] After running, the chip was scanned in a Sensospot fluorescence scanner under green light to illuminate the Cy3 eluted primers that had hybridized to the amplicon generated on the chip. Based on the fluorescence intensity, the amplification on the chip could be qualitatively determined.

[0114] result

[0115] The detection of two assays was quantitatively established using the voltage output of a δISFET sensor. Liquid-phase PCR amplification was qualitatively evaluated using E-gel analysis and Sensospot fluorescence imaging of the chip surface.

[0116] Example 1: Single-target anchored primer amplification and detection (LAPAD)

[0117] Target: Serratia marcescens

[0118] Chip ID: NM-248-0214

[0119] The anchoring primer sequences used in Example 1 can be found in Table 4.

[0120] Table 4: Anchoring primer sequences used in Example 1

[0121]

[0122] The average delta signal of an ISFET can be found in [reference]. Figure 11 .

[0123] For the δ signal of all ISFETs with anchored primer target positives, please refer to [link to relevant documentation]. Figure 12 .

[0124] For the δ signal of all ISFETs with anchored primer target negative, please refer to [link to relevant documentation]. Figure 13 .

[0125] For the complete ISFET δ signal of the anchored primer target control, please refer to [link to relevant documentation]. Figure 14 .

[0126] For the average ISFET signal results of positive, negative, and control primers, please refer to [link to relevant documentation]. Figure 15 For the positive anchor primer NH2_iSp18_SmR, an average ISFET signal of 36.2 dmV was detected, which is even greater than the average ISFET signal of 23.4 dmV for the control anchor primer NH2_ddl_5'+T15.

[0127] Following PCR, the liquid-phase amplicon generated by *Serratia marcescens* is shown in the gel electrophoresis image. Figure 16 For the positive anchoring primer NH2-iSp18_SmR, the generated amplicon on the chip can be viewed along with the control anchoring primer in the sensospot fluorescence image. Figure 17).

[0128] The amplification and detection of Serratia marcescens in a single target region were confirmed. A significant δISFET voltage signal was detected, which was even greater than that of the control anchor primer.

[0129] Example 2: Multiplexed Targeted Amplification and Detection (LAPAD)

[0130] Target: Schizosaccharomyces cerevisiae

[0131] Chip ID: NM-248-0172

[0132] The anchoring primer sequences used in Example 2 can be found in Table 5.

[0133] Table 5: Anchoring primer sequences used in Example 2.

[0134]

[0135] For the average δ signal of the ISFET in Example 2, please refer to... Figure 18 .

[0136] For the δ signal of all ISFETs with anchored primer targets 1 to 4 positive, please refer to [link to relevant documentation]. Figure 19 .

[0137] For the δ signal of all ISFETs with anchored primer negative, please refer to [link to relevant documentation]. Figure 20 .

[0138] For the complete ISFET δ signal of the anchored primer control, please refer to [link to relevant documentation]. Figure 21 .

[0139] For the average ISFET signal results of positive, negative, and control primers, please refer to [link to relevant documentation]. Figure 22 For the four positive anchor primers, ISFET signals ranging from 1.5 dmV to 4.4 dmV were detected. This is significantly lower than the average ISFET signal of the control anchor primer, which had a previous signal intensity of 34.6 dmV.

[0140] Following PCR, the liquid phase amplicon generated by *Schizosaccharomyces cerevisiae* is shown in the gel electrophoresis image. Figure 23 On the Sensospot fluorescence image, for the positive anchor primer, the generated amplicon on the chip could not be observed. Figure 24 However, control anchoring primers can be observed.

[0141] The amplification and detection of *Schizosaccharomyces cerevisiae* in multiple target regions were confirmed.

Claims

1. A method for amplifying and analyzing nucleic acid sequences, the method being used for non-disease diagnostic purposes, the method comprising: a. A system having a liquid portion, wherein the liquid portion comprises a nucleic acid sample, two or more immobilized amplification primers and reagents for nucleic acid amplification, and one or more immobilized amplification primers; wherein the immobilized amplification primers are in excess relative to the immobilized amplification primers; b. Perform an asymmetric nucleic acid amplification reaction using the aforementioned non-immobilized amplification primers to produce a first non-immobilized nucleic acid amplification product; c. Further amplify the first non-immobilized nucleic acid amplification product using one or more of the immobilized amplification primers to generate an immobilized second nucleic acid amplification product; d. Real-time query of the localization signal generated at the immobilized second nucleic acid amplification product, and e. Detect the presence, absence, or sequence of the second nucleic acid amplification product.

2. The method according to claim 1, wherein the ratio of the non-immobilized amplification primer to the immobilized amplification primer is 3:

1.

3. The method of claim 1, wherein additional primers are released or added during the amplification reaction or at a predetermined time point.

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

5. The method according to claim 1, wherein the amplification product is subjected to optical detection.

6. The method of claim 1, wherein the detection of the amplification product comprises a fluorescent reporter probe and a subsequent fluorescence assay.

7. The method according to claim 1, wherein the immobilized amplification primer amplifies the internal segment of the first non-immobilized nucleic acid amplification product, such that the immobilized second nucleic acid amplification product is shorter than the first non-immobilized nucleic acid amplification product.

8. The method of claim 1, wherein the liquid portion is exposed to two or more immobilized amplification primers, wherein each primer amplifies a different sequence.

9. The method of claim 8, wherein the two or more immobilized amplification primers differ by a single base, thereby detecting a single-base variant in the first non-immobilized nucleic acid amplification product.

10. The method of claim 1, wherein the method is performed on an array-type optical imaging system, the array-type optical imaging system including a surface in contact with the liquid portion.

Images