Combined liquid phase and solid phase DNA amplification

Abstract

The application relates to combined liquid phase and solid phase DNA amplification. Methods for efficient amplification and detection of certain nucleic acid sequences in a population are described. The selected population may be further characterized (e.g., by sequencing). The method includes combined liquid phase 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 name “Combined liquid and solid phase DNA amplification”. Technical Field

[0002] The present invention relates to methods for sequence-specific amplification of a subpopulation of nucleic acid fragments from a population of a wider range of nucleic acid sequences. The present invention specifically detects only the desired target sequence. Aspects of the present invention relate to nucleic acid constructs for use in such methods. The present invention describes methods for effectively targeting amplification and detecting specific nucleic acid sequences in a population. Background Art

[0003] Identification of pathogens in blood and other biological samples is essential for the effective treatment of a variety of disease states, including sepsis. In the UK, 5 people die every hour from sepsis, and 25% of all sepsis survivors suffer permanent, life-changing consequences. Early detection and identification of the pathogens of bacterial infection is essential to preventing the onset of sepsis and successfully treating sepsis. Existing methods for detecting and identifying blood-borne infections include blood culture and the use of antibiotic sensitivity testing. These methods involve the culture of cells, which is expensive both in time and money. Often, septic shock will occur before cell culture results are available.

[0004] The use of existing molecular detection methods such as PCR that can identify pathogens in samples by analyzing the nucleic acid of the pathogen is limited by low overall sensitivity levels. This is particularly a problem 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 the target (pathogen) nucleic acids, leading to false negative results, or give false positive readings of pathogen nucleic acids due to non-specific amplification.

[0005] For example, there are several antibiotic resistance genes that have multiple variants due to point mutations or other polymorphisms, and the presence of a specific mutation can lead to a different treatment strategy than 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 laboratories can use sequencing to elucidate internal sequences and identify polymorphisms - which is time-consuming and expensive to implement. Therefore, the selective amplification of a population of target nucleic acids relative to non-target nucleic acids is of great value to the pharmaceutical industry and research.

[0006] Amplified reaction usually depends 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 the cross hybridization between the primers, it is usually not feasible to combine a large number of primer pairs. Therefore, usually use the independent primers in the independent solution part to carry out multiple PCR, thereby need a large amount of samples. The present invention describes the improvement of amplified reaction, and the improvement of this amplified reaction can carry out sequence selective amplification to multiple different primers in a single sample amount.

[0007] Also disclosed is a method for emulsion PCR, wherein a single template is diluted so that the bubbles in the oil emulsion contain less than one template molecule per bubble on average. A bead with a single immobilized primer may be included, with a second primer in solution. Thus, a mixture of two primers is used for amplification, wherein one primer is immobilized and the other primer is in solution. However, in contrast to the further amplification of the first amplification product in the methods described herein, only a single amplification product is produced in each reaction portion.

[0008] Amplification methods are also known in which both primers are immobilized, such as the bridge amplification based on the Illumina cluster. In this case, there are no primers in free solution. Summary of the invention

[0009] The invention provides a two-phase amplification reaction, including a (thermal cycle or isothermal) amplification reaction of both solid phase and liquid phase, which can use a combination of immobilized and non-immobilized primers and target DNA templates to generate DNA amplicons in solution and on a surface simultaneously. The presence or absence of the amplicon can be detected in an endpoint test after amplification or in real time. This two-phase method is different from the prior art including emulsion PCR because it includes solid phase (i.e. surface-based) amplification and liquid phase amplification.

[0010] The nucleic acid sample can be amplified using one or more non-immobilized primers. The liquid phase generated DNA and target DNA template can also serve as or subsequently serve 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. The readout provides further discrimination capabilities for the system by fixing primers of different sequences at known positions and using a detection system that can discern the positions (e.g., using ISFETs on CMOS IC chips or arrays based on fluorescence / optical imaging systems).

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

[0012] The present application describes a method, including a method for amplifying and analyzing a nucleic acid sequence, the method comprising:

[0013] a. contacting a system having a liquid volume with a solid support, wherein the liquid volume comprises a nucleic acid sample, two or more non-immobilized amplification primers and a reagent for nucleic acid amplification, and the solid support has one or more immobilized amplification primers;

[0014] b. performing a nucleic acid amplification reaction using a non-immobilized amplification primer to produce a first non-immobilized nucleic acid amplification product;

[0015] 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;

[0016] d. One of the following

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

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

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

[0020] 2. flowing a solution comprising at least one nucleotide over the immobilized second amplification product to extend the hybridized primer, and

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

[0022] The method can be performed as follows, wherein the non-immobilized amplification primers in the liquid portion are released from the solid phase prior to amplification. The nucleic acid amplification reaction using non-immobilized primers can use two pairs or more non-immobilized primers. Nucleic acid amplification at any stage can be isothermal, or nucleic acid amplification at any stage can be performed by thermal cycling.

[0023] According to the method, the primers hybridized to the second nucleic acid amplification product may be fluorescently labeled and subsequent fluorescence measurement may be achieved. Alternatively, the method may be performed in which the amplification product is detected by, for example, an ISFET sensor by detecting protons released upon nucleotide incorporation.

[0024] The method can be performed in such a way that a first amplification product and a second amplification product are obtained using amplification primers having the same nucleic acid sequence.

[0025] The method may also include amplifying an internal segment of the first amplification product with an immobilized amplification primer such that the second amplification product is shorter than the first amplification product.

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

[0027] The immobilized primers of the method can be located on an open microarray or in separate wells. The separate wells can be exposed to the same reagent portion simultaneously. If the wells contain immobilized primers with different sequences, different amplicons can be generated in different wells, thereby enabling multiple amplification within a single liquid portion.

[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 support, wherein the liquid portion contains a nucleic acid sample, two or more non-immobilized amplification primers and a reagent for nucleic acid amplification, and the solid support has one or more immobilized amplification primers, wherein the system includes a sensor for detecting the amplification product.

[0029] The amplification product can be detected by optical or electrical methods. For optical detection, the amplification product needs to be labeled with a fluorescent marker, which can then be detected by an optical scheme.

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

[0031] Figure 1 : Schematic diagram of single position anchor primer amplification and detection (LAPAD) is shown.

[0032] Figure 2 : Schematic diagram of multiplex localization anchor primer amplification and detection (LAPAD) is shown.

[0033] Figure 3: shows a schematic diagram of performing elution. It occurs on an open system requiring on-chip flow capabilities. During the amplification reaction, the product amplicons in the liquid phase fall onto the solid phase primers, which are extended to produce many 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 enzymes are loaded onto the chip, and after a short hybridization step, all four nucleotides flow in together, producing a burst of protons on those ISFETs with extended oligonucleotides. This proton burst is very clearly observed in the most simplified data processing.

[0034] Figure 4 The elution workflow and the data obtained therefrom are shown. The first row represents the amplification phase of the experiment, producing a cluster of extended amplicons on the chip surface. This is followed by a dehybridisation step, for example using heating, to produce single-stranded amplicons on the chip surface, with no product in the liquid phase. After the elution primer and enzyme hybridization, all four nucleotides flow through the chip and a signal is generated on those ISFETs that have been specifically extended.

[0035] Figure 5 Sample ISFET data is shown showing the elution signals generated by the three target templates after PCR. Data shown are for E. coli (80 bases of product, so a maximum of 60 bases eluted), E. faecalis, and S. marcescens after specific amplification of the relevant targets. Data was not modified, all ISFETs are shown.

[0036] Figure 6 A multiplex chip spotted with three anchor primers and a positive template control is shown. PCR was performed with the Ef template followed by elution. The heat map shows the ISFET voltage measured ~15 seconds after nucleotide flow. It can be seen that the ISFETs match the spotted pattern and correspond to both the Ef and positive controls. The trace on the right shows an example of the ISFET response for each ISFET group.

[0037] Figure 7A sequencing implementation is shown. Instead of adding all four nucleotides for elution, the nucleotides are added sequentially. This occurs on an open system that requires on-chip flow capabilities. During the amplification reaction, the product amplicon in the liquid phase lands on the solid phase primer, which is extended to produce many extended amplicons on the chip surface. After amplification, the amplification product 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 a burst of protons on those ISFETs that have the amplification product and the correct template base. Single-base resolution can be achieved.

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

[0039] Fig. 9 Real-time detection workflow is shown. Can occur on a platform without fluid flow or with minimal fluid flow after chip amplification. Can react in one or more reaction chambers that are permanently sealed. During amplification reaction, the product amplicon in the liquid phase falls on the solid phase oligomer, and the specific oligomer is modified and extended. In a sealed system with low buffering, during the extension phase of each cycle of amplification reaction, protons will be generated near the solid phase. Promoting asymmetry to produce a significant excess of forward primers in the liquid phase will effectively generate a "micro-elution" burst of protons in each cycle, which should be detectable in subsequent cycles. As an alternative to primer asymmetry when the amplification reaction begins, additional primers can be released or added during the amplification reaction or at a set time point. Amplification reaction will be carried out in a low buffer to be able to detect pH signals in real time.

[0040] Fig.10 Data for real-time detection are shown. The chip was spotted in batches with multiple anchored primers, PCR (3:1 asymmetric) with 6 chamber gaskets (3 chambers fluid sealed), eluted in a flow station with a single chamber. The liquid phase shows a real-time pH signal, indicating that the reaction is ongoing. As confirmation of post-PCR amplification, a fluorescent tag targeting the distal end of the amplicon was used as a probe for the chip and showed that the anchored primers were extended during the PCR reaction.

[0041] Fig.11 The average delta signal for the ISFET of Example 1 is shown.

[0042] Fig.12 The delta signal of all ISFETs positive for the anchor primer target of Example 1 is shown.

[0043] Fig.13 The delta signal for all ISFETs negative for the anchor primer target of Example 1 is shown.

[0044] Fig.14 The delta signal for all ISFETs for the anchor primer control of Example 1 is shown.

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

[0046] Fig.16 1 is a gel electrophoresis diagram showing the generation of liquid phase amplicons after PCR for Serratia marcescens of Example 1.

[0047] Fig.17 Sensospot fluorescence images for Example 1 are shown.

[0048] Fig.18 The average delta signal for the ISFET of Example 2 is shown.

[0049] Fig.19 Delta signals positive for anchor primer targets 1 to 4 of Example 2 are shown.

[0050] Fig. 20 The delta signal for all ISFETs negative for the anchor primer of Example 2 is shown.

[0051] Fig.21 The delta signal for all ISFETs for the anchor primer control of Example 2 is shown.

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

[0053] Fig.23 This is the gel electrophoresis image for Example 2.

[0054] Fig.24 This is the Sensospot fluorescence image for Example 2. DETAILED DESCRIPTION

[0055] This application describes a system having the ability to generate reads of different sequences from the same reaction portion.

[0056] In brief, the system includes a two-phase amplification reaction, wherein a liquid phase (PCR or isothermal) amplification reaction occurs in the presence of an immobilized primer on a solid phase that further participates in producing 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 a solution, and when enough products have been produced, the system detects a corresponding signal. The nucleic acid amplification product generated by this liquid phase and the target nucleic acid template can also serve as or subsequently serve as a template for an additional reaction performed on the solid phase, whereby, as part of the amplification reaction, one or more immobilized primers interact with the liquid phase template and generate a nucleic acid sequence that can be subsequently detected by the system. By fixing the primers at known positions and using a detection system that can distinguish the position (e.g., using an ISFET on a CMOS IC chip or an array-type optical imaging system), the resulting reading enables selective amplification and detection of many amplicons in the same device.

[0057] In the presence of one or more other primers fixed on the solid phase, an amplification reaction is performed in the liquid phase. The immobilized primer can be the same sequence as one or more liquid phase primers, or it can be an internal sequence corresponding to the nucleic acid template generated in the liquid phase. The sequence identical to one or more liquid phase primers can be called a second time by the same reaction component, and the internal sequence corresponding to the nucleic acid template generated in the liquid phase is called a second time, which increases the confirmation and improves the confidence, that is, the correct amplification reaction product has been generated and / or further provides information on the internal sequence of the target template.

[0058] 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 custom systems that integrate real-time detection of both liquid and solid phase reactions. The repeatability potential of ISFET CMOS allows for a large number of repetitions of each test, as well as multiple target genes for each target, thereby increasing the confidence of the system by relying on multiple reads for each target. With this design, it is also advantageous to incorporate positive and negative controls in each reaction vessel, thereby ensuring correct adjudication when controls are performed as needed.

[0059] Below are different embodiments of combining liquid and solid phase amplification reactions.

[0060] DNA amplification readout

[0061] The immobilized primer can be identical or similar to one or more liquid phase primers, and participates in amplified reaction, so that compared with the amplification carried out in the absence of liquid phase primers, the signal generated by the immobilized primer increases, thereby increasing the confidence of the result, but does not further add information to the sequence generated. For example, by making the primer consumed in the liquid phase be present on the solid phase, the asymmetric PCR in the liquid phase can be compensated. Along with the carrying out of amplified reaction, the liquid phase product generated is prompted to interact with the solid phase, thereby generating liquid phase amplicon and solid phase amplicon. Immobilized amplicon can be detected. There is this reading on a plurality of positions of fixed primers, so that the confidence that the material amplified subsequently is clearly present in the sample increases. If the immobilized primer with different sequences can not give a positive result, the confidence of this result increases so.

[0062] DNA amplification with internal confirmation

[0063] The immobilized primers can correspond to the internal sequence of the DNA template generated in the liquid phase, so that any signal generated on the solid phase is a positive confirmation, that is, 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 (for example, when the target of interest is a pathogen in a host sample, a signal generated by interacting with host (human) DNA) and provides significant benefits relative to standard confirmation tests such as melting curve analysis and electrophoresis gel imaging because other sequence information is inferred in the readout.

[0064] Internal confirmation signals are similar to standard laboratory confirmation procedures such as melting curve analysis, electrophoresis gel imaging, etc.

[0065] The use of immobilized primers corresponding to internal sequences in the target template allows further specificity to be imparted to the system, whereby the combination of liquid phase and solid phase reactions identifies more sequence information than liquid phase reactions alone, thereby increasing the confidence in the results compared to liquid phase alone.

[0066] DNA amplification with internal sequence information

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

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

[0069] In this embodiment, a single primer pair can be used in the liquid phase to amplify all variants of a gene, wherein one or more immobilized primers corresponding to a point mutation or polymorphism can be used on the solid phase to identify one or more variants. The combination of each internally immobilized primer provides the user with rich information of the overall sequence of the amplified gene and can make clinical decisions easier and faster.

[0070] Elution (including multiple elutions)

[0071] Amplification reactions are usually carried out in high ionic strength buffers. For sequencing systems that measure the proton release of the incorporated nucleotides, if the amplification buffer is replaced with a buffer with low buffering capacity, the amplification product can be detected. Alternatively, fluorescently labeled primers or via the use of fluorescently labeled nucleotides can be used to detect the amplification product.

[0072] Elution occurs on an open system requiring on-chip flow capabilities. During the amplification reaction, the product amplicons in the liquid phase land on the solid phase primers, which are extended to produce many 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 enzymes are loaded onto the chip, and after a short hybridization step, all four nucleotides flow together, producing a burst of protons on those ISFETs with extended oligonucleotides. This proton burst is very clearly observed in the most simplified data processing.

[0073] In order to perform electron / proton detection, at the end of the aforementioned amplification phase with both liquid and solid phase reactions, the liquid phase component is flushed out of the column and replaced with a solution with low buffering capacity. Then, primers and enzymes or a combination of primers and enzymes are sequentially followed by nucleotide streams, which will release protons when nucleotides are incorporated, and corresponding signals are generated on the detection platform, such as mV changes on the relevant ISFET. The primers used for elution can correspond to any position in the amplification template, including universal sequences added during early amplification reactions. Using amplicon-specific primers inside the solution, the amplification product provides increased confidence in the results by confirming that the correct substance has been produced on the solid phase, but due to having a higher multiplicity, the complexity of the system may be increased. If universal sequences are added in early amplification reactions, the scale of liquid phase multiple amplification may be limited, thereby improving the efficiency of the reaction. However, since universal sequences may have misfiring, this may limit the reading confidence.

[0074] By knowing the location of the specific immobilized primer, the identity of the target can be determined when the protons generated during the elution reaction produce a signal on the specific ISFET sensor to which the specific primer is immobilized. In this embodiment, multiple replicates of each assay target are possible, with the scale of the multiplex being limited by the spatial separation, number of wells and sensors per device, and bioinformatics challenges.

[0075] The system can utilize a single elution reaction or multiple elution reactions, with or without a dehybridization step between each run. Additional elutions can be used to query earlier signals so that confidence in the internal sequence can be increased. For example, if a universal sequence is used in an early amplification reaction, the first elution can use the universal sequence to identify which experiments have generated signals, followed by elutions using specific primers corresponding to those specific experiments to confirm that the correct product has been generated.

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

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

[0078] At the end of the above-mentioned amplification phase, the liquid phase component can be flushed out of the column and replaced with a solution with low buffering, as the elution workflow.Then, it is a combination of primer and enzyme or primer and enzyme, followed by a single nucleotide stream or a combination stream of two or more nucleotides, when the correct complementary nucleotide flows through the chip and mixes, a proton will be released.This workflow provides the most abundant information of all embodiments, because it provides sequence information, rather than relying only on the comparison of primers and their corresponding templates.This workflow not only confirms that correct comparison has occurred and correct product has been produced on solid phase, but also indicates internal sequence by inference or by direct identification.As discussed in the elution embodiment, similar methods can be used herein, wherein multiple operations can provide further information, and the workflow of this method combination of elution and one or more nucleotides used.

[0079] Real-time detection

[0080] By measuring the extension of the immobilized primers, the amplification process can be monitored in real time. The reaction can be carried out in one or more reaction chambers that are permanently sealed. During the amplification reaction, the product amplicons in the liquid phase fall onto the solid phase oligomers, which are modified and extended for the specific oligomers. In a sealed system with low buffering, protons will be generated in close proximity to 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, wherein the two primers in the solution are present at different concentrations, producing a predominantly single-stranded amplification product due to the lack of complementary primers for their extension. Driving asymmetry to produce a significant excess of forward primers in the liquid phase will effectively generate a "micro-elution" burst of protons in each cycle, which should be detectable in subsequent 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 a set time point. The amplification reaction will be carried out in a low buffer to be able to detect pH signals in real time.

[0082] 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 represent a fragment of a genome; for example, a single chromosome or a single genomic locus (e.g., for rapid sequencing of allelic polymorphisms). In a particular embodiment, the amplification of the 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 cDNA copy thereof.

[0083] Before carrying out the claimed amplification reaction, biological substances can be amplified in solution. Therefore, before carrying out the present invention, the substance may have undergone a sequence-based amplification step. Alternatively, the sample can be processed to connect the universal sequence shared by one or both ends of all chains of the sample. The universal sequence can be used as a universal sequence that hybridizes with a common primer that may be non-natural.

[0084] The invention described herein can allow for four or more levels of amplification specificity. A first amplification can be performed in solution. A second amplification can be performed using a non-immobilized primer. A third amplification can be performed using an immobilized primer that is different in sequence from the second amplification primer. The presence of the amplicon can be detected using primer specificity for the amplicon generated by the immobilized primer. Thus, four levels of amplification specificity can be used to ensure that the final amplicon is detected only when the starting sequence is absolutely and unequivocally present in the sample. Thus, false positive results from detection of closely related but undesirable sequences can be eliminated.

[0085] It is conventional to fix nucleic acid on the surface. Any method of covalent or non-covalent immobilization can be used. Ideally, for multiple cycles of thermal cycling to the temperature causing the separation of nucleic acid chains, immobilization is stable. The specific method of immobilization includes UV-induced crosslinking or immobilization via formation of amide bonds or phosphorothioate bonds.

[0086] Example

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

[0088] Schematic diagrams of single LAPAD and multiple LAPAD are shown in Figure 1 and Figure 2 shown.

[0089] Example 1: Single Positioning Anchor Primer Amplification and Detection (LAPAD)

[0090] Amplification and detection of Serratia marcescens at a single target region. The target region at the 3′ end of the amplification template DNA hybridizes with a complementary anchor primer target. During the amplification process, the anchor primer target is extended, and in the next stage, the same target region is detected during the extension. The primer / oligonucleotide sequences used are shown in Table 1.

[0091] Table 1: Oligonucleotide sequences

[0092]

[0093] Example 2: Multiple Positioning Anchor Primer Amplification and Detection (LAPAD)

[0094] Amplification and detection of Schizosaccharomyces pombe at multiple target regions.

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

[0096] Experimental methods

[0097] The '004'Ta composed of ISFETs will be combined 2 O 5 The chip platform of the CMOS chip and enclosed in SU-8 wells is anchored with primers using UV-based cross-linking 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 detection DNA, and the PCR reagent recipe, 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 formula

[0100]

[0101] Table 3: Thermal Cycling Conditions

[0102]

[0103] After PCR, the chip was washed with wash buffer (0.06x saline-sodium citrate, 0.06% Tween 20), and then the DNA was chemically dehybridized with 20 mM NaOH for 5 minutes and then washed again with wash 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) primer was 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 with the eluted primer mixture was then heated at 95°C for 2 minutes, then at 58°C for 5 minutes, and incubated at room temperature for 15 minutes. 0.06% Tween 20) to wash the chip.

[0106] An enzyme mix was then added to the surface of the chip, which was created 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] The incorporation of deoxyribonucleotide triphosphates (dNTPs) was achieved by using a custom flow system that enables dNTPs to flow on the chip surface. 12.5 μM of each 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 pH of the dNTP mixture was adjusted to pH 8 and maintained by keeping the fluid under nitrogen.

[0108] ISFET sensor detection

[0109] Spot the raw ISFET sensor voltage output data and anchor primers Figure 1 The ISFET voltage signal peak height is detected and the anchor primer ISFET voltage signal peak height is subtracted from the voltage signal peak height of the adjacent upstream blank ISFET. This is done to remove system noise from the fluid stream, including noise that may mask the detection results. This delta ISFET sensor voltage output of all specific anchor primer sensors is averaged to determine the signal detection result.

[0110] E-gel electrophoresis analysis

[0111] Prepared E-gel (48-well 2% agarose gel stained with ethidium bromide) was used for qualitative analysis of 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 into the E-gel wells. Gradient loading was also performed to compare the size of the amplicons. The gel was run for 10 minutes on an E-gel substrate that provided an electric field to allow the DNA amplicons to migrate according to their size.

[0112] Sensospot fluorescence imaging

[0113] After the run, the chip is scanned in a Sensospot fluorescence scanner under green light to illuminate the Cy3 eluted primers that have hybridized to the amplicons generated on the chip. Based on the fluorescence intensity, the amplification on the chip can be qualitatively determined.

[0114] result

[0115] Detection of both assays was established quantitatively by delta ISFET sensor voltage output. Liquid-phase PCR amplification was qualitatively evaluated by E-gel analysis and chip surface amplification by Sensospot fluorescence imaging.

[0116] Example 1: Single Positioning Anchor Primer Amplification and Detection (LAPAD)

[0117] Target: Serratia marcescens

[0118] Chip ID: NM-248-0214

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

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

[0121]

[0122] The average delta signal of the ISFET can be found in Fig.11 .

[0123] The delta signal for all ISFETs positive for the anchor primer target can be found in Fig.12 .

[0124] The delta signal for all ISFETs negative for the anchor primer target can be found in Fig.13 .

[0125] The delta signal for all ISFETs for the anchor primer target control can be found in Fig.14 .

[0126] The average ISFET signal results for positive, negative and control primers can be found in Fig.15 For the positive anchor primer NH2_iSp18_SmR, an average ISFET signal of 36.2 dmV was detected, which was even greater than the average ISFET signal of 23.4 dmV for the control anchor primer NH2_ddl_5′+T15.

[0127] After PCR, the liquid phase amplicon generated by Serratia marcescens is shown in the gel electrophoresis image ( Fig.16 For the positive anchor primer NH2-iSp18_SmR, the generated on-chip amplicon can be seen in the sensor spot fluorescence image together with the control anchor primer ( Fig.17).

[0128] Amplification and detection of Serratia marcescens at a single target region was confirmed. A clear delta ISFET voltage signal was detected, which was even greater than that of the control anchor primer.

[0129] Example 2: Multiple Positioning Anchor Primer Amplification and Detection (LAPAD)

[0130] Target: Schizosaccharomyces pombe

[0131] Chip ID: NM-248-0172

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

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

[0134]

[0135] The average delta signal of the ISFET of Example 2 can be found in Fig.18 .

[0136] The delta signals for all ISFETs positive for anchor primer targets 1 to 4 can be found in Fig.19 .

[0137] The delta signal for all ISFETs negative for the anchor primer can be found in Fig. 20 .

[0138] The delta signal for all ISFETs for the anchor primer control can be found in Fig.21 .

[0139] The average ISFET signal results for positive, negative and control primers can be found in Fig. 22 ISFET signals ranging from 1.5 dmV to 4.4 dmV were detected for the four positive anchor primers. This is much lower than the previous signal intensity and the average ISFET signal of the control anchor primer of 34.6 dmV.

[0140] After PCR, the liquid phase amplicons generated by S. pombe are shown in the gel electrophoresis image ( Fig.23 ). In the Sensospot fluorescence image, for the positive anchor primer, the generated on-chip amplicon cannot be observed ( Fig.24 ). However, the control anchor primer can be observed.

[0141] Amplification and detection of Schizosaccharomyces pombe in multiple target regions were confirmed.

Claims

1. A method for multiplex sequence-specific amplification of a subpopulation of nucleic acid fragments from a broader population of nucleic acid sequences, comprising: contacting a system having a liquid portion with a solid support, wherein the liquid portion comprises a nucleic acid sample, two or more pairs of non-immobilized amplification primers, and a reagent for nucleic acid amplification, wherein the solid support has two or more immobilized amplification primers configured to amplify two or more different sequences, wherein the amplification primers of the different sequences are immobilized at known positions, and wherein the system includes a detection system capable of discerning the positions of the immobilized primers; a. performing a nucleic acid amplification reaction using the non-immobilized amplification primer to produce a first non-immobilized nucleic acid amplification product; b. further amplifying the first non-immobilized nucleic acid amplification product using the two or more immobilized amplification primers to produce an immobilized second nucleic acid amplification product; One of the following i. real-time query of the localization signal generated at the immobilized second nucleic acid amplification product, or ii. removing the liquid portion and the first non-immobilized amplification product and replacing it with a new liquid portion having primers that hybridize to the immobilized second nucleic acid amplification product, and one of the following directly detecting the hybridization primer; or flowing a solution comprising at least one nucleotide over the immobilized second amplification product to extend the hybridized primer, and The presence, absence or sequence of the second nucleic acid amplification product is detected.

2. The method of claim 1, wherein the immobilized primers are located on an open microarray or in individual wells.

3. The method according to claim 1 or 2, wherein the immobilized primers are located in separate wells and the separate wells are exposed to the same reagent portion simultaneously.

4. The method according to any one of claims 1 to 3, wherein the non-immobilized amplification primers in the liquid portion are released from the solid phase prior to amplification.

5. The method according to any one of the preceding claims, wherein the amplification is performed by thermal cycling.

6. The method according to any one of claims 1 to 4, wherein the amplification is isothermal amplification.

7. The method according to any one of claims 1 to 6, wherein the detection of the immobilized second nucleic acid amplification product is performed by interrogating the generated localization signal in real time.

8. The method of claim 1, wherein the two primers in the solution are present at different concentrations to produce asymmetric amplification.

9. The method according to any one of claims 1 to 8, wherein the primer hybridizing to the second nucleic acid amplification product is fluorescently labeled.

10. The method according to any one of claims 1 to 8, wherein the amplification product is detected by detecting protons released upon nucleotide incorporation.

11. The method of claim 10, wherein the detecting uses an ISFET sensor.

12. The method according to any one of claims 1 to 8, wherein the detection of the amplification product comprises a fluorescent reporter probe and subsequent fluorescence measurement.

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

14. The method according to any one of the preceding claims, wherein the immobilized amplification primer amplifies an internal segment of the first amplification product such that the second amplification product is shorter than the first amplification product.

15. The method according to any one of the preceding claims, wherein the two or more immobilized amplification primers differ by a single base, thereby detecting a single base variant in the first nucleic acid amplification product.

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