Method for multiplex isothermal amplification of nucleic acid sequences - Patents.com

Abstract

A method is disclosed for the isothermal amplification of nucleic acid molecules, optionally on a solid support, that uses a single-stranded nucleic acid, or an extension product thereof, having common / universal hairpin regions at both the 3' and 5' ends.

Description

[Technical field]

[0001] Methods for isothermal amplification of nucleic acids are disclosed. Constructs, methods and kits are disclosed that enable a form of loop-mediated amplification that can be performed either in solution or on a solid support. [Background technology]

[0002] Loop-mediated isothermal amplification (LAMP) is a technique first reported by Notomi et al. in 2000 (Nucleic Acids Res. 2000;28(12):E 63), and is a rapid and efficient method for isothermal amplification of nucleic acids. This technique uses a strand-displacing DNA polymerase (typically Bst polymerase) and a set of four specific oligonucleotide primers designed to six different regions of the target template DNA, and typically achieves approximately 10 fold amplification with a small template input within a short reaction time (e.g., 60 min at 65°C). 9 It can be faithfully amplified to the point of copying.

[0003] Due to its sensitivity, specificity, and rapid time to results, LAMP has been used as the basis for numerous diagnostic assays for a variety of infections and diseases. Being an isothermal method means that it can be used in the field without the need for sophisticated thermal cycling equipment, making it ideally suited for point-of-care applications.

[0004] Methods for next generation nucleic acid sequencing can involve amplifying multiple sequences in parallel, often on a population of solid supports or beads such that strands of different sequences are amplified in physical isolation, a so-called clonal amplification.

[0005] LAMP has several features that make it attractive as a clonal amplification method: Rapid amplification; it is paramount to generate multiple copies of the input template as quickly as possible. The absolute copy number per parent template molecule is also key to maximizing the detection signal. Isothermal method; this simplifies the workflow and cartridge function of the components. No sophisticated thermal cycling hardware is required to run the assay. High fidelity; ensures that the amplification produces multiple features that, when sequenced, reflect as closely as possible the identity of the starting template. Low-complexity reaction; few reaction components are required to support amplification, ensuring robust and reproducible performance. Low cost of goods; the method is inexpensive to implement.

[0006] However, conventional LAMP has several drawbacks that make it less attractive as a clonal amplification method:

[0007] Multiplexing samples and loci in a single tube reaction is difficult and complicated. Because each amplicon requires a unique set of four primers, LAMP reactions are typically limited to singleplex or duplex assays. Increasing the plex of a single reaction increases the risk of mispriming and off-target amplification. The only practical way to multiplex traditional LAMP is to run multiple different singleplex reactions in parallel.

[0008] Typically, the progress of LAMP amplification or the products of the LAMP reaction are detected by real-time or end-point methods (typically turbidity or increase in fluorescence due to amplification). Although LAMP products are amenable to DNA sequencing techniques due to their low multiplexing capacity and their typical use in low-cost assays, their use in combination with DNA sequencing is limited to confirmation of correct amplification and does not use DNA sequencing itself as a diagnostic readout.

[0009] The products of LAMP amplification are high molecular weight structures formed from several inverted repeat concatemeric copies of the patent template strand. These can be linear or more complex branched cauliflower-like structures, depending on the reaction conditions.

[0010] A variety of LAMP amplification detection methods exist, but these tend to rely on real-time or end-point solution amplification product detection. There have been no previous reports of the use of LAMP to generate surface-bound isothermal amplification.

[0011] The present inventors have devised a means to enable loop-mediated amplification both in solution and on a solid support. Summary of the Invention

[0012] The present inventors have developed an improved isothermal amplification method that works both in solution and on a surface, making it amenable as a clonal amplification method, and in so doing retains many of the beneficial features and improves many of the negative features encountered in solution-LAMP isothermal amplification.

[0013] The method of the present invention relies on single-stranded nucleic acid molecules engineered to contain hairpin regions at both the 3' and 5' ends. The 3' end of the hairpin can undergo polymerase extension, thereby creating a double-stranded material with one end that is an "open" blunt end linked by a central hairpin. Thus, the material to be amplified is a single-stranded molecule under fully denaturing conditions with both sense and antisense strands linked to each other via a central adapter that also has adapters at both the 5' and 3' ends, with the adapters at the ends being complementary.

[0014] In the double-stranded form, the 3' end is blunt-ended and therefore has no template. In the single-stranded form, the ends are self-complementary and therefore can self-prime. Amplification is performed using a primer that extends half of the strand by hybridizing to the central loop. The first primer extension thereby gives material that is half double-stranded and half single-stranded. The single-stranded 3' end can self-prime, thereby generating a molecule that replicates the double-stranded product. The double-stranded concatemer has one end that is an "open" blunt end connected by a central hairpin that also has a central known double-stranded adapter region.

[0015] Hybridization of the primer to the central loop and its extension again produces a molecule in which half the strand is single-stranded and half is double-stranded. Again, the 3' end of the single-stranded region can self-anneal and extend. In this way, concatemer amplification can be achieved using a primer complementary to the single-stranded loop and self-extension of the released 3'-end. With each "cycle", the molecule effectively doubles the length of the 3' end produced by the polymerase, as the displaced strand can generate a copy of the entire strand.

[0016] As described herein, clonal amplification can be achieved by immobilizing one of the primers on a solid support. [Brief description of the drawings]

[0017] [Figure 1] Figure 3 shows a general LAMPlate structure. The molecule has a single-stranded central portion flanked by hairpins at both the 3' and 5' ends. The 3' end can be extended to arrive at the construct shown in Figure 4. The extension can be an initial stage of amplification or can be carried out as a separate "pre-amplification" step. [Diagram 2] FIG. 1 shows how LAMPlates can be generated simply by simultaneously ligating LP1 and LP2 adapters to a single-stranded template molecule. [Diagram 3] FIG. 1 shows how LAMPlates can be generated by PCR by using locus-specific PCR primers with 5′ stem-loop structures. [Figure 4] FIG. 4 shows a LAMPlate after intramolecular self-priming extension from an internal 3' end. Extension opens the hairpin via strand displacement to reach the 5' end of the sequence without the need for an additional primer. Extension can be an early stage of amplification, such that amplification is initiated using the construct of FIG. 1, or can be performed as a separate "pre-amplification" step, in which case the amplified material is shown in FIG. 4. [Diagram 5] Illustrates the extension of the hybridized first primer into the internal single-stranded loop (primer LP1, hybridization with LP1). After extension, the molecule is half single-stranded and half double-stranded. The displaced 3'-end has a hairpin stem that can self-prime (LP2'), thereby creating a concatemer and displacing the extended primer. The displaced extended primer also has a self-complementary end that can self-prime (also LP2'). Thus, the first primer produces a "single" copy and a double copy of the double-stranded template, each with a blunt end and a closed loop end (LP2'). Both central loops are single-stranded regions of the same sequence. [Figure 6]Illustrates the extension of the hybridized second primer into the newly formed internal single-stranded loop (primer P2, hybridization to LP2'). After primer extension, the molecule is again half single-stranded and half double-stranded. The displaced 3'-end has a hairpin stem that can self-prime (again LP2'), thereby creating a concatemer and displacing the extended primer. The displaced extension primer also has a self-complementary end that can self-prime. Thus, the first primer produces a "single" copy and a double copy of the double-stranded template, each with a blunt end and a closed loop end (LP2'). Both central loops are single-stranded regions of the same sequence (LP2'), and the loops have the same sequence as the loop from the first stage of amplification. The P2 primer can repeatedly hybridize and extend, thereby creating multiple copies of increasing length. [Figure 7] The equivalent of Figure 4, where only the amplification primer LP1 is immobilized. Both the self-primed 3' end of the LAMPlate and the 3' end of the immobilized primer can undergo extension. The extended material takes the form of a double-stranded and a single-stranded portion (Figure 7). [Figure 8] The single-stranded portion has a self-priming 3'-end that can undergo extension and thereby remove itself from the solid support into solution (Figure 8). [Figure 9] The displaced material is "dead" and no complementary primer is present on the support, however, the displacement frees the 3' end of the immobilized product, which can self-prime and extend to make the immobilized material fully double-stranded from the central loop (Figure 9). [Figure 10]10 shows displacement using a solution-phase primer hybridized to an immobilized loop. The primer hybridized to the loop can be extended, thereby displacing the 3' end of the immobilized molecule. The displaced 3' end can self-prime and thereby be extended. However, the displaced 3' end also releases a free single-stranded loop that is complementary to the immobilized primer. The loop can hybridize to a local primer, thereby extending a local copy of itself (FIG. 10). [Figure 11] Similar to the single and double loops in solution, each amplicon on the surface now has a single and double loop localized within the length of the immobilized nucleic acid (Figure 11). The extension product from the non-immobilized primer can form a double-stranded molecule in solution. The immobilized primer LP1 can hybridize to the loop, thereby allowing further amplification that is spatially localized within the reach of the immobilized template. Thus, the strand can be locally amplified. [Figure 12] Similar to the single and double loops in solution, each amplicon on the surface now has a single and double loop localized within the length of the immobilized nucleic acid (Figure 11). The extension product from the non-immobilized primer can form a double-stranded molecule in solution. The immobilized primer LP1 can hybridize to the loop, thereby allowing further amplification that is spatially localized within the reach of the immobilized template. Thus, the strand can be locally amplified. [Figure 13]Non-denaturing agarose gel images show INS01 LAMPLate ligation products, with the trend of molecular weight shift from adapter only, mono-adapter INS01 and dual-adapter INS01 clearly observable. Lanes 1 and 13 are molecular weight ladders. Lane 2 is insert only. Lanes 3-6 are adapter pairs LP1 and LP2 of two designs (v1 and v2) and two concentrations (either 280 pmol L3, 4 or 660 pmol L5, 6 per adapter). Lanes 7-10 are mono-adapter ligations. Lanes 11-12 are full ligations for v1 and v2 adapter designs. [Figure 14] Non-denaturing agarose gel images show five individual locus-specific PhiX-174 PCR amplicons using looped PCR primers, with clean bands indicating amplification of products of expected molecular weight. Lanes 1 and 7 are molecular weight ladders. Lanes 2-6 are amplicons for regions LAMP_PhiX_01-05. The 5' loops of the PCR_F and PCR_R primers add an additional 103 nt to the insert length and thus the expected shift in molecular weight over the expected amplicon insert length. [Figure 15] The non-denaturing agarose gel image shows a band pattern typical of LAMP amplified DNA. A ladder of bands of increasing molecular weight is consistent with tandem repeat concatemer amplification indicative of this method. Lanes 1 and 6 are molecular weight ladders. Lanes 2-3 and 4-5 are LAMPlicons generated by solution LAMP amplification of 1E8 and 1E10 copies of full-length Ultramer LAMPlate LAMP_T01_v1, respectively. [Figure 16]Solution LAMP amplification of single LAMPlates prepared by ligation. The non-denaturing agarose gel image shows a band pattern typical for LAMP amplification of various single LAMPlicon constructs. The gel clearly shows the specificity of the amplification. Only the complete double-ligated LAMPlate supports the amplification. The V1 adapter design seems to be superior to the v2 design. Lanes 1, 15 are molecular weight ladders. Lane 2 is only the SHRK_INS01 insert subjected to a solution LAMP amplification reaction. The adapter pairs in lanes 3, 4 (v1 and v2, respectively) were subjected to a solution LAMP amplification reaction. Lanes 5, 6 are the v1 mono-adapter insert subjected to a solution LAMP amplification reaction. Lanes 7, 8 are the v2 mono-adapter insert subjected to a solution LAMP amplification reaction. Lanes 9, 10 are the complete double-adapter LAMPlates (v1 and v2, respectively) subjected to a solution LAMP amplification reaction. Lanes 11 and 12 are no ligase controls for full duplex adaptor LAMPlates (v1 and v2, respectively) subjected to solution LAMP amplification reactions. Lanes 13 and 14 are full duplex adaptor LAMPlates (v1 and v2, respectively) subjected to solution LAMP amplification reactions using incorrect solution primers (v2 and v1 primers, respectively). [Figure 17]Solution LAMP amplification of single LAMPlates prepared by ligation. The non-denaturing agarose gel image shows a pattern of bands typical for LAMP amplified DNA with various constructs. The gel clearly shows the specificity of the amplification. Only complete doubly ligated LAMPlates support amplification. Lanes 1, 18, 19 and 30 are molecular weight ladders. Lanes 2-8, 20-21 are ssDNA inserts alone subjected to solution LAMP amplification reactions. Lanes 9-16 are serial dilutions of 7-plex ligation-generated LAMPlate pools (1E7, 1E8 and 1E9 copies input) subjected to solution LAMP amplification reactions. Lanes 22, 23 are adapters alone subjected to solution LAMP amplification reactions. Lanes 24, 25 are LP1 mono-adapter inserts (inserts SBL_T01 and SBL-T02, respectively) subjected to solution LAMP amplification reactions. Lanes 26 and 27 are LP2 monoadapter inserts (inserts SBL_T01 and SBL-T02, respectively) subjected to solution LAMP amplification reactions. Lanes 28 and 29 are no-ligase controls (inserts SBL_T01 and SBL-T02, respectively) subjected to solution LAMP amplification reactions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] By adding hairpin loops (termed LP1 and LP2) to the ends of the target template DNA fragments, library molecules capable of supporting LAMP amplification, referred to herein as "LAMPlates", are generated. A general LAMPlate structure is shown in FIG. 1. This pair of hairpin adaptors can be the same for all LAMPlate library molecules in a given pool and are therefore universal adaptors. This feature allows all library molecules in a pool, regardless of the insert sequence, to be interrogated and amplified with a common set of primers specific for these universal hairpin adaptors. This aspect of the invention provides a route to in situ amplification of highly multiplexed LAMPlate molecules in a single-pot reaction, something that is difficult or impossible to achieve with standard isothermal LAMP amplification.

[0019] The DNA sequence of the universal adaptor is platform specific and can be easily tailored to the requirements. The only constraints are that the stem region of each adaptor needs to be a complementary DNA duplex and the loop region connects the two arms of the stem and must have a different sequence than the stem to prevent mishybridization. The length and GC content of the stem arm may be important for the efficient formation of the intramolecular hairpin loop and thus the efficiency of the resulting amplification. The length of the stem arm may be between 15 and 30 bp long and have a Tm ranging from 45 to 55 °C.

[0020] The length and GC content of the loop may be important to achieve efficient hybridization of the amplification primer and therefore to the efficiency and specificity of the resulting amplification. The loop length is likely to be between 15-30 bp in length and have a Tm (when annealed to its complementary amplification primer) in the range of 55-65°C. It is necessary that the DNA sequences of the LP1 and LP2 hairpin adapters are sufficiently different from each other to promote the specificity and efficiency of the resulting LAMPlate amplification. The adapter pair is expected to add approximately 90-180 nt of sequence to the target insert.

[0021] The stem duplex can be designed to encode one or more restriction or nicking endonuclease motifs that may be useful for post-amplification processing of the amplified LAMPlate.

[0022] It is anticipated that there is no limit to the length of the insert that can be adapted, but for practical purposes, inserts of size 50-200 nucleotides are likely to be optimal for LAMPlate amplification efficiency, and inserts longer than 1000 nucleotides are unlikely to be required.

[0023] LAMPlates can be generated simply by co-ligating LP1 and LP2 adapters to a single-stranded template molecule (Figure 2) or by PCR by using a locus-specific PCR primer with a 5' stem-loop structure (Figure 3).

[0024] The template is suitable for amplification to form concatemers. Amplification is performed with a strand-displacing polymerase. The hairpin stem is self-complementary, and once displaced by the polymerase to become single stranded, the released single-stranded 3'-end can self-anneal and be extended. Thus, amplification can effectively be performed using the copied molecule as its own template.

[0025] Disclosed is a population of single-stranded nucleic acid molecules (ss1) of at least 50 nucleotides in length, where the single strands have different sequences, each of the single strands having a common hairpin region (LP1) at its 3' end and a common hairpin region (LP2) at its 5' end, each hairpin having a hybridizing stem arm 15-30 base pairs in length, the stem arms having a Tm in the range of 45-55°C.

[0026] The source single-stranded 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, such as a single chromosome or a single genomic locus (e.g., for rapid sequencing of allelic polymorphisms). In certain instances, the amplification may be specific to a pathogenic agent within the sample. For example, the amplification may select for bacterial or viral nucleic acids present within a human sample. The template may be DNA, RNA, or a cDNA copy thereof.

[0027] The viral nucleic acid may be derived from a coronavirus, a group of related RNA viruses that cause disease in mammals and birds. They cause respiratory tract infections such as the common cold, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as COVID-19.

[0028] SARS-CoV-2 is the strain of coronavirus responsible for the global pandemic COVID-19, which is a positive-sense single-stranded RNA virus. Each SARS-CoV-2 virus is 50-200 nanometers in diameter and has four structural proteins known as the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The N protein carries the RNA genome, while the S, E and M proteins together form the viral envelope.

[0029] Thus, in one embodiment, the amplification may select for one or more viral nucleic acids encoding each structural protein of SARS-CoV-2.

[0030] In one embodiment, the amplification can select for one or more nucleic acids, the presence of which is indicative of an active SARS-CoV-2 infection in the host. In one embodiment, the amplification can select for one or more nucleic acids, the presence of which is indicative of a previous SARS-CoV-2 infection in the host. These nucleic acids can be nucleic acids produced by the host as part of an immune response to SARS-CoV-2 infection.

[0031] In one embodiment, each single-stranded nucleic acid molecule (ss1) has a single-stranded loop (L) between the stem arms, the loop being 15-30 nucleotides in length and having a Tm in the range of 55-65°C when annealed to a complementary primer.

[0032] In one embodiment, the single-stranded nucleic acid molecule is 50 to 200 nucleotides in length. In one embodiment, the single-stranded nucleic acid molecule is 100 to 200 nucleotides in length.

[0033] In one embodiment, the single-stranded nucleic acid molecules of the population have a total length of 190-380 and a double-stranded sequence of 15-30 base pairs at the 3' end and 5' end at a temperature of less than 45°C.

[0034] A method for concatemeric amplification of a plurality of nucleic acid sequences is disclosed, the method comprising: taking a population of nucleic acid molecules according to any one of the preceding embodiments of the invention and a reaction mixture containing a strand-displacing polymerase, nucleotide monomers and amplification primers, at least one of the amplification primers (P1') being complementary to at least a portion of the loop (L) of LP1 and one of the amplification primers (P2) comprising a sequence identical to at least a portion of a copy of the loop (LP2') of LP2.

[0035] A method for concatemeric amplification of a plurality of nucleic acid sequences is disclosed, the method comprising: a) obtaining a population of single stranded nucleic acid molecules (ss1) differing in length by at least 50 nucleotides, the single strands having different sequences, each of the single strands having a common hairpin region (LP1) at its 3' end and a common hairpin region (LP2) at its 5' end, each hairpin having a hybridization stem arm of 15-30 base pairs in length, the stem arm having a Tm in the range of 45-55°C; b) amplifying a population of distinct single-stranded nucleic acid molecules (ss1) using a reaction mixture containing a strand-displacing polymerase, nucleotide monomers and amplification primers, at least one of the amplification primers (P1') being complementary to at least a portion of the loop (L) of LP1 and one of the amplification primers (P2) containing a sequence identical to at least a portion of a copy of the loop of LP2 (LP2'); Includes.

[0036] In one embodiment of the method, the 3' end of the stem arm is extended to create a complete copy of the single stranded nucleic acid molecule at least 50 nucleotides long (ss1') and a complete copy of sequence LP2 (referred to as LP2'). The extension can be the first part of the amplification reaction. Alternatively, the 3' end of the stem arm can be extended to create a complete copy of the single stranded nucleic acid molecule at least 50 nucleotides long (ss1') and a complete copy of sequence LP2 (referred to as LP2'), and then the extended molecule can be placed into an amplification reaction mixture.

[0037] In a further embodiment of the method, P1' hybridizes to a loop of LP1 and strand displaces, thereby extending until it separates LP2 and LP2'. Strand displacement generates complementary copies of ss' and LP2' (i.e., a replica of ss and a replica of LP2).

[0038] In a further embodiment of the method, the stem arm at the end of LP2' can self-prime and extend to create complete copies of all of ss1', LP1, ss1 and LP2.

[0039] In a further embodiment of the method, amplification primer P2 hybridizes to the loop of LP2' and is extended to displace all of ssl', LP1, ssl and LP2.

[0040] In a further embodiment of the method, the released end LP2' is capable of repeatedly self-priming to copy the concatemers ss1', LP1 and ss1.

[0041] In a further embodiment of the method, the amplification primer P1 is attached to a solid support.

[0042] In a further embodiment of the method, the amplification primer P2 is in solution.

[0043] It is envisioned that the amplification methods may be used to generate surface-bound amplification products, particularly clonal surface-bound amplification products, according to the schemes depicted in Figures 7-12.

[0044] A DNA oligo primer, called LP1RC, whose sequence is complementary to the LP1 loop of the LAMPlate, is immobilized on a solid support (e.g., the surface of a slide, chip or bead). This functionalized surface can be incubated at a specified temperature for a specified time in the presence of a solution containing a dilute concentration of LAMPlate (e.g., 1-1000 pM) in an appropriate hybridization buffer. This allows the LAMPlate to anneal to the immobilized primer via sequence complementarity with the single-stranded LAMPlate LP1 loop region. This method can be called "LAMPlate seeding".

[0045] Once seeded, excess LAMPlate can be washed away, leaving only the surface primer-hybridized molecules in place. Addition of a strand-displacing DNA polymerase (e.g. Bst or Phi29 polymerase) and dNTPs in an appropriate reaction buffer allows extension from the free 3' end of the LAMPlate and simultaneously from the free 3' end of the surface primer hybridized to the LAMPlate loop LP1. This surface primer extension causes displacement of the extended LAMPlate duplex, thus forming a molecule that is partly single-stranded and partly double-stranded and immobilized via the 5' end of the shorter strand.

[0046] Thus, the extended 3' end of the LAMPlate is single-stranded and capable of self-priming. The self-primed end can extend to make a perfect copy, in the process displacing itself from the immobilized strand. The material displaced thereby into the bulk solution is completely self-complementary apart from a single loop, which is of a different sequence than the loop hybridized to the surface, and therefore the material in the bulk solution plays no further role in the amplification.

[0047] This displacement causes the spontaneous reformation of an intramolecular LP2' loop at the 3' end of the immobilized copy, which can self-prime and extend to generate a surface-bound duplex with an LP2' hairpin distal to the surface. Thus, the immobilized material is mostly double-stranded, except for one internal loop, LP2'.

[0048] If necessary, the fluid reagents can be changed at this point to remove the displaced material, thereby preventing further amplification of this material, which could contaminate the amplification method or simply consume amplification reagents.

[0049] Amplification on the surface requires a non-immobilized primer that is complementary to the immobilized single-stranded loop (i.e., a primer that hybridizes to LP2'). A solution oligo-primer, called LP2, that is complementary to the LP2' loop of the immobilized duplex product, can anneal to the LP2' loop and be extended. This LP2 primer extension forms a faithful copy of one half of the immobilized duplex and simultaneously displaces the 3' end of the strand that is complementary to the copy strand. The displaced 3' end spontaneously reforms the LP1 intramolecular hairpin that can both self-prime and hybridize to further copies of the immobilized primer LP1RC via the single-stranded loop. Thus, two new extendable 3' ends are available.

[0050] The process of forming new propagated interactions by cross-linking one strand from one copy to an adjacent surface primer is called "template walking". Subsequent simultaneous duplex extension from the free 3' end of the hybridized LP1 loop and from the free 3' end of the newly formed duplex between the LP1 loop of the walked template strand and the second LP1RC surface primer allows the generation of two surface-immobilized hairpin duplexes via extension, strand displacement and self-primed complementary extension. The first duplex is a duplex concatemer of the initial LAMPlate with the LP1 loop distal from the surface (i.e., four consecutive duplex sets of ss and ss'), and the second duplex is a duplex with the hairpin loop LP1 distal from the surface and a single concatemer (i.e., two repeats of ss and ss') (shown in FIG. 12).

[0051] Both immobilized duplexes have a single-stranded loop complementary to the LP1RC surface primer. Thus, the surface primer can hybridize to the center of the immobilized strand. Extension displaces the immobilized 3' end with the loop LP1. Reformation of LP1 gives a self-priming end and a loop that can be immobilized to further copies of primer LP1RC. This results in the formation of two new extendable 3' ends, one self-priming and one on the immobilized primer. Thus, amplification is self-sustaining through cycles of hybridization to the support, causing displacement and self-priming extension.

[0052] This sequential method of primer hybridization, extension, displacement and template walking continues for the duration of the amplification reaction or until the reagents in the reaction are consumed, whichever occurs first. The product of these amplification and template walking cycles is the generation of multiple faithful surface-bound copies of the original seeded LAMPlate as intramolecular concatemeric repeat duplexes in spatial proximity to the original seeded LAMPlate. Multiple such closely spaced faithful duplex copies can be referred to as clones, or clusters or colonies, and thus the original single-stranded portion of the seeded LAMPlate can be said to be "clonally amplified".

[0053] Methods for determining the presence, absence or sequence of the nucleic acid amplification products of the invention are also disclosed.

[0054] Also disclosed is a device for the amplification of a plurality of nucleic acid sequences comprising a solid support having immobilized amplification primers hybridized to a population of nucleic acid molecules described herein.

[0055] Also disclosed is a kit for amplifying a plurality of nucleic acid sequences, the kit comprising: a. a first hairpin adapter having hybridization stem arms 15-30 base pairs in length, the stem arms having a Tm in the range of 45-55° C. and a single-stranded loop (L) between the stem arms, the loop being 15-30 nucleotides in length and having a Tm in the range of 55-65° C. when annealed to a complementary primer; b. a second hairpin adapter having a hybridization stem arm of 15-30 base pairs in length, the stem arm having a Tm in the range of 45-55° C. and a single-stranded loop (L) between the stem arms, the loop being 15-30 nucleotides in length and having a Tm in the range of 55-65° C. when annealed to a complementary primer; c. a solid support having an immobilized amplification primer, the primer being complementary to at least a portion of the loop L of the first hairpin adaptor; D. Nucleic acid polymerase and nucleoside triphosphates Equipped with.

[0056] method Preparation of LAMPlate by ligation (A69_SBLAMP_E001) material -LP1 oligo (LAMP_LP1_v1) -LP2 oligo (LAMP_LP2_v1) -Template oligo (INS01) -T4 DNA ligase 2000U / uL(NEB,M0202T) -Zymo Oligo Clean&Concentrate Purification Column -EB buffer (10mM Tris.HCl pH8.0)

[0057] [Table 1]

[0058] method Typically, ligation reactions were prepared by mixing LP1 and LP2 adapter oligos in equimolar ratios (280 pmol of each adapter) with template oligo INS01 (130 pmol) in 1× ligation buffer. Reactions were mixed briefly by vortexing. The resulting oligos were heat denatured at 95°C for 3 min and then annealed by a slow cooling ramp (-0.1°C / sec to 15°C) on a thermocycler. Ligation reactions were initiated by the addition of 2000U T4 DNA ligase (1 uL of 2000U / uL T4 ligase stock), mixed by brief vortexing, and then incubated at 15°C for 30 min. The ligase was inactivated by heat sterilization at 75°C for 5 min and purified using Zymo Oligo Clean&Concentrate spin columns. Final elution was in EB buffer. Samples were run on a 3% TBE agarose gel stained with Gel Red stain for visualization.

[0059] Legend for Figure 13 Non-denaturing agarose gel images show INS01 LAMPLate ligation products, with the trend of molecular weight shift from adapter only, mono-adapter INS01 and dual-adapter INS01 clearly observable. Lanes 1 and 13 are molecular weight ladders. Lane 2 is insert only. Lanes 3-6 are adapter pairs LP1 and LP2 of two designs (v1 and v2) and two concentrations (either 280 pmol L3, 4 or 660 pmol L5, 6 per adapter). Lanes 7-10 are mono-adapter ligations. Lanes 11-12 are full ligations for v1 and v2 adapter designs.

[0060] Preparation of LAMPlate by loop PCR (A69_SBLAMP_E022) material -Looped PCR_F primer -Looped PCR_R primer -Template genomic DNA (bacteriophage PhiX-174, NEB, N3022L) -PCR buffer (5X buffer components, Thermo F122S) -Phire II HS DNA polymerase (polymerase component, Thermo F122S) -10mM dNTP mix -Qiagen PCR Purification Columns -EB buffer (10mM Tris.HCl pH8.0)

[0061] [Table 2]

[0062] method Individual PCR amplification reactions were prepared for each PhiX amplicon using the SBL_PHIX PCR primer pair. Each 50 uL PCR reaction consisted of 10 ng PhiX-174 genomic DNA, 20 pmol each of PCR_F and PCR_R primers, 10 nmol dNTP mix, 1 uL Phire II HS DNA polymerase in 1X Phire PCR buffer. The reaction mixture was denatured at 98°C for 2 minutes and then subjected to 25 subsequent cycles of denaturation (95°C for 30 seconds), annealing (60°C for 20 seconds) and extension (72°C for 20 seconds). Amplicons were purified using Qiagen PCR cleanup columns and the final eluate was placed in EB buffer. Samples were run on a 3% TBE agarose gel stained with Gel Red stain for visualization.

[0063] Legend for Figure 14 Non-denaturing agarose gel images show five individual locus-specific PhiX-174 PCR amplicons using looped PCR primers, with clean bands indicating amplification of products of expected molecular weight. Lanes 1 and 7 are molecular weight ladders. Lanes 2-6 are amplicons for regions LAMP_PhiX_01-05. The 5' loops of the PCR_F and PCR_R primers add an additional 103 nt to the insert length and thus the expected shift in molecular weight over the expected amplicon insert length.

[0064] Solution-LAMP amplification using full-length Ultramer LAMPlate oligos material -LAMPlate Ultramer template oligo (LAMP_T01_v1) - Solution LAMP amplification primers (LP1rc_v1 and LP2_v1) -Warm Start LAMP Amplification Kit (NEB, E1700S) -Zymo Oligo Clean&Concentrate Purification Column -EB buffer (10mM Tris.HCl pH8.0)

[0065] [Table 3]

[0066] method Example 2 5uL solution LAMP amplification reactions were prepared by diluting 12.5uL of 2X LAMP master mix (containing buffer, Bst 2.0 Warm Start DNA polymerase and dNTPs) with Ultramer LAMPlate LAMP_T01_v1 (0.25-2.5 fmol), LAMP primers LP1rc_INV_v1 (230 pmol) and LP2_INV_v1 (230 pmol) and MilliQ water. The reaction was initiated by warming to 65°C and incubating the reaction for 60 minutes. The reaction was stopped by the addition of 3uL of 500mM EDTA or directly purified by Qiagen PCR cleanup columns. Samples were run on a 3% TBE agarose gel stained with Gel Red stain for visualization.

[0067] Legend for Figure 15 The non-denaturing agarose gel image shows a band pattern typical of LAMP amplified DNA. A ladder of bands of increasing molecular weight is consistent with tandem repeat concatemer amplification indicative of this method. Lanes 1 and 6 are molecular weight ladders. Lanes 2-3 and 4-5 are LAMPlicons generated by solution LAMP amplification of 1E8 and 1E10 copies of full-length Ultramer LAMPlate LAMP_T01_v1, respectively.

[0068] Solution LAMP amplification using a single ligation preparation LAMPlate material - Purified ligation preparation LAMPlate (SHRK_INS01) -LAMP adapter (LP1_v1, LP2_v1, LP1_v2, LP2_v2) - Solution LAMP amplification primers (LP1rc_v1, LP2_v1, LP1rc_v2, LP2_v2) -Warm Start LAMP Amplification Kit (NEB, E1700S) -Zymo Oligo Clean&Concentrate Purification Column -EB buffer (10mM Tris.HCl pH8.0)

[0069] [Table 4]

[0070] method Example 2 5uL solution LAMP amplification reactions were prepared by diluting 12.5uL of 2X LAMP master mix (containing buffer, Bst2.0 Warm Start DNA polymerase and dNTPs) with individual ligation-prepared LAMPlates (using either v1 or v2 adapter designs and insert SHRK_INS 01 as described in Example 1), LAMP primers LP1rc_INV_v* (230pmol, *=v1 or v2) and LP2_INV_v* (230pmol, *=v1 or v2) and MilliQ water. A range of conditions were tested as outlined in Figure 4. Reactions were initiated by warming to 65°C and incubating the reactions for 60 minutes. Reactions were stopped by the addition of 3uL of 500mM EDTA or directly purified by Qiagen PCR clean-up columns. Samples were run on a 3% TBE agarose gel stained with Gel Red stain for visualization.

[0071] Legend to Figure 16: Solution LAMP amplification of a single LAMPlate prepared by ligation. The non-denaturing agarose gel image shows a band pattern typical of LAMP amplification of various single LAMPlicon constructs. The gel clearly shows the specificity of the amplification. Only the complete double-ligated LAMPlate supports the amplification. The V1 adapter design appears to be superior to the v2 design. Lanes 1 and 15 are molecular weight ladders. Lane 2 is only the SHRK_INS01 insert subjected to a solution LAMP amplification reaction. The adapter pairs in lanes 3 and 4 (v1 and v2, respectively) were subjected to a solution LAMP amplification reaction. Lanes 5 and 6 are the v1 mono-adapter insert subjected to a solution LAMP amplification reaction. Lanes 7 and 8 are the v2 mono-adapter insert subjected to a solution LAMP amplification reaction. Lanes 9 and 10 are the complete double-adapter LAMPlate (v1 and v2, respectively) subjected to a solution LAMP amplification reaction. Lanes 11 and 12 are the no-ligase controls for the complete double-adapter LAMPlate (v1 and v2, respectively) subjected to a solution LAMP amplification reaction. Lanes 13, 14 are full duplex adaptor LAMPlates (v1 and v2, respectively) that were subjected to solution LAMP amplification reactions using incorrect solution primers (v2 and v1 primers, respectively).

[0072] Solution LAMP Amplification using Multiple Ligation Prepared LAMPlate Pools material -LAMPlate insert (SBL_T01-7) -LAMP adapter (LP1_v1 and LP2_v1) - Solution LAMP amplification primers (LP1rc_v1 and LP2_v1) -Warm Start LAMP Amplification Kit (NEB, E1700S) -Qiagen PCR Purification Columns -EB buffer (10mM Tris.HCl pH8.0)

[0073] [Table 5]

[0074] method Example 2 A 5 uL solution LAMP amplification reaction was prepared by diluting 12.5 uL of 2X LAMP master mix (containing buffer, Bst2.0 Warm Start DNA polymerase and dNTPs) with ligation-prepared LAMPlate pool (using LP1_v1 and LP2_v1 adapters and pool of all seven inserts SBL_T01-7 as described in Example 1), LAMP primers LP1rc_INV_v1 (230 pmol) and LP2_INV_v1 (230 pmol) and MilliQ water. A range of conditions were tested as outlined in Figure 4. Reactions were initiated by warming to 65°C and incubating the reactions for 60 minutes. Reactions were stopped by the addition of 3 uL of 500 mM EDTA or directly purified by Qiagen PCR cleanup column. Samples were run on a 3% TBE agarose gel stained with Gel Red stain for visualization.

[0075] Legend to Figure 17: Solution LAMP amplification of a single LAMPlate prepared by ligation. The non-denaturing agarose gel image shows a band pattern typical for LAMP amplified DNA with various constructs. The gel clearly shows the specificity of the amplification. Only the complete double-ligated LAMPlate supports amplification. Lanes 1, 18, 19 and 30 are molecular weight ladders. Lanes 2-8, 20-21 are ssDNA inserts alone subjected to solution LAMP amplification reactions. Lanes 9-16 are serial dilutions of 7-plex ligation-generated LAMPlate pools (1E7, 1E8 and 1E9 copies input) subjected to solution LAMP amplification reactions. Lanes 22, 23 are adapters alone subjected to solution LAMP amplification reactions. Lanes 24, 25 are LP1 mono-adapter inserts (inserts SBL_T01 and SBL-T02, respectively) subjected to solution LAMP amplification reactions. Lanes 26, 27 are LP2 mono-adapter inserts (inserts SBL_T01 and SBL-T02, respectively) subjected to solution LAMP amplification reactions. Lanes 28, 29 are no-ligase controls subjected to solution LAMP amplification reactions (inserts SBL_T01 and SBL-T02, respectively). [Sequence List Free Text]

[0076] Sequence Listings 1 and 3 <223> T at position 1 has PO4 at position 5 Sequence Listing 5 <223> G at position 1 has PO4 at position 5 Sequence Listings 6, 8, 10, 12, 14 <223> The G, A, C, and T at positions 1, 2, 3, and 4 are linked by phosphorothioate bonds. Sequence Listing 17 <223> The C, G, A and G at positions 34, 35, 36 and 37 are linked by phosphorothioate bonds Sequence Listing 18 <223> C, C, A and C at positions 30, 31, 32 and 33 are linked by phosphorothioate bonds Sequence Listing 19 <223> The T, C, T and G at positions 29, 30, 31 and 32 are linked by phosphorothioate bonds Sequence Listing 20 <223> The G, T, G and G at positions 29, 30, 31 and 32 are linked by phosphorothioate bonds

Claims

1. A method for surface-bound concatemeric amplification of a plurality of nucleic acid sequences, comprising: a) obtaining a population of single stranded nucleic acid molecules (ss1) differing in length by at least 50 nucleotides; wherein said single strands have different sequences, each of said single strands has a common hairpin region (LP1) at its 3' end and a common hairpin region (LP2) at its 5' end, each hairpin having a hybridization stem arm 15-30 base pairs in length, said stem arms having a Tm in the range of 45-55° C.; and b) amplifying a population of distinct single-stranded nucleic acid molecules (ss1) using a reaction mixture containing a strand-displacing polymerase, nucleotide monomers and amplification primers, wherein at least one of the amplification primers (P1') is bound to a solid support and is complementary to at least a portion of the loop (L) of LP1 and one of the amplification primers (P2) contains a sequence identical to at least a portion of a copy of the loop (LP2') of LP2.

2. The method of claim 1, comprising: i) annealing primer P1' to the loop of LP1; ii) extending the 3' end of the stem arm of LP1 to create an exact copy of a single stranded nucleic acid molecule of at least 50 nucleotides in length (ss1') and an exact copy of the sequence of LP2 (LP2'), where P1' extends until it strand displaces, thereby separating LP2 and LP2'; iii) self-priming and extending the stem arm at the end of LP2', creating a complete copy of all of ssl', LP1, ssl and LP2, thereby releasing it from the immobilized strand; iv) extending amplification primer P2 to hybridize to loop LP2' and displace all of ssl', LP1, ssl and LP2, copying one half of the immobilized dimer and displacing the 3' end of the strand complementary to the copied strand; v) allowing the displaced 3′ end of the strand complementary to the copied strand to reform hairpin LP1; allowing hairpin LP1 to self-prime and hybridize to a second primer P1′ that is attached to the solid support via a single-stranded loop; and vi) allowing extension from the free 3' end of the hybridized loop LP1 and the free 3' end of the newly formed duplex between loop LP1 and the second primer P1' to generate two surface-immobilized hairpin duplexes.

3. The method of claim 1 or 2, wherein the surface-bound primer P1 further comprises the sequence of the 3' stem of loop LP1.

4. The method of claim 3, wherein the surface-bound primer P1 comprises, in the 5' to 3' direction, the sequence of the 3' stem of loop LP1 and a sequence complementary to the loop (L) of LP1.

5. The method of claim 2, wherein the fluid reagent is changed after step iii).

6. The method according to any one of claims 1 to 5, wherein the released end LP2' is capable of repeatedly self-priming to copy the concatemers ss1', LP1 and ss1.

7. The method according to any one of claims 1 to 6, wherein the presence, absence or sequence of a nucleic acid amplification product is determined.

8. A kit for amplifying a plurality of nucleic acid sequences comprising: a. a first hairpin adaptor having a hybridization stem arm 15-30 base pairs in length, said stem arm having a Tm in the range of 45-55° C. and a single-stranded loop (L) between said stem arms, said loop being 15-30 nucleotides in length and having a Tm in the range of 55-65° C. when annealed to a complementary primer; b. a second hairpin adaptor having a hybridization stem arm 15-30 base pairs in length, said stem arm having a Tm in the range of 45-55° C. and a single-stranded loop (L) between said stem arms, said loop being 15-30 nucleotides in length and having a Tm in the range of 55-65° C. when annealed to a complementary primer; c. a solid support having an immobilized amplification primer, said primer being complementary to at least a portion of loop L of said first hairpin adaptor; d. a primer that contains a sequence identical to at least a portion of a copy of the single-stranded loop of the first hairpin adaptor and the loop of the common hairpin region at the 5' end of the single-stranded loop of the second hairpin adaptor; and e. Nucleic acid polymerase and nucleoside triphosphates.

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