Nucleic Acid and Gene Synthesis

Abstract

The present invention relates to a method for synthesizing nucleic acids, and more particularly, to a method for extending overlapping primer oligonucleotides to generate oligonucleotides, polynucleotides, gene fragments or genes having repetitive sequences. The present invention also relates to overlapping primer oligonucleotides for use in nucleic acid synthesis reactions.

Description

[Technical Field]

[0001] The present invention relates to a method for synthesizing nucleic acids. The present invention also relates to overlapping primer oligonucleotides for use in nucleic acid synthesis reactions. [Background technology]

[0002] Nucleic acid synthesis is the process of linking nucleotides to form nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids can be synthesized using a variety of techniques, including phosphoramidite synthesis, slippage reaction, loop-mediated amplification (LAMP), primer-template extension, and thermocycling amplification.

[0003] The synthesis of nucleic acids containing repetitive sequences is challenging using existing nucleic acid synthesis methods, especially for long sequences (over 200 base pairs (bp)). Challenges in synthesizing longer nucleic acid sequences typically stem from decreased yield and quality as longer sequences are generated. One of the most commonly used methods for nucleic acid synthesis is column-based oligonucleotide synthesis, which uses phosphoramidite synthesis chemistry to sequentially add bases to a growing oligonucleotide sequence on a solid support. This method utilizes a four-step process—deblocking (detritylation), coupling, capping, and oxidation—that is repeated to build the oligonucleotide sequence. Upon completion of synthesis, the oligonucleotide is chemically cleaved from the solid support. This process is particularly amenable to automation and is the basis for many commercial gene synthesis systems. A major drawback of column-based oligonucleotide synthesis is that the yield and quality of the oligonucleotide product decrease with sequence length. This is due to spurious depurination, particularly at adenosines, during synthesis. These spurious sites promote cleavage of the oligonucleotide backbone, resulting in decreased yield. This decrease in yield and quality means that this method is not suitable for producing oligonucleotides longer than 200 bases. Phosphoramidite synthesis is also inefficient for producing sequences with high GC content, hairpin structures, and / or highly repetitive sequences.

[0004] Alternatively, enzymatic techniques can be used to synthesize nucleic acids. These methods typically have fewer limitations on sequence length compared to phosphoramidite synthesis. Enzymatic techniques use nucleic acid templates or primers and polymerase enzymes to synthesize nucleic acid sequences by polymerase chain reaction (PCR). One such example is polymerase cycle assembly (PCA). PCA uses PCR to extend short overlapping oligonucleotides into double-stranded sequences. Enzymatic synthesis techniques are suitable for generating longer sequences, but are inefficient at generating highly repetitive sequences and sequences with high GC content.

[0005] Because long nucleic acid sequences with multiple repeat units have many applications in synthetic biology, diagnostics, and therapeutics, there is a need to identify more efficient methods for their production. Summary of the Invention

[0006] According to a first aspect of the present invention, (i) providing at least one overlapping primer oligonucleotide, the overlapping primer oligonucleotide comprising a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, each of the first single-stranded oligonucleotide and the second single-stranded oligonucleotide comprising at least a first, a second, and a third sequence, the third sequence being located at a 3' end of each single-stranded oligonucleotide, the second sequence being located between the first sequence and the third sequence, and the first sequence and the third sequence of the first single-stranded oligonucleotide and the second single-stranded oligonucleotide being self-complementary palindromic sequences; the third sequence of the first single-stranded oligonucleotide hybridizes to the third sequence of the second single-stranded oligonucleotide to provide an overhang region at the 5' end of at least one of the first or second single-stranded oligonucleotide; Steps and (ii) enzymatically extending both the first single-stranded oligonucleotide and the second single-stranded oligonucleotide to form a duplex; The present invention provides a method for extending the length of a nucleic acid, comprising:

[0007] Advantageously, the extension step (ii) incorporates a fourth sequence into the first single-stranded oligonucleotide, said fourth sequence being complementary to a second sequence on the second single-stranded oligonucleotide, and a fourth sequence into the second single-stranded oligonucleotide, said fourth sequence being complementary to a second sequence on the first single-stranded oligonucleotide.

[0008] Advantageously, the present invention provides improved methods for synthesizing nucleic acids, in particular, methods that provide a more efficient means for generating longer nucleic acid sequences that can be designed according to user requirements.

[0009] Furthermore, this method provides an improved means for synthesizing long repetitive sequences that are difficult to synthesize using existing methods due to low yields and high error rates.

[0010] Advantageously, having a 3' self-complementary sequence ensures that the sequence will be predictably incorporated into the oligonucleotide during extension, allowing the user more control over the order of sequences in the nucleotide product.

[0011] Typically, a fourth sequence is incorporated into the first and second single-stranded oligonucleotides during step (ii), the fourth sequence on the first single-stranded oligonucleotide being complementary to the second sequence on the second single-stranded oligonucleotide, and the fourth sequence on the second single-stranded oligonucleotide being complementary to the second sequence on the first single-stranded oligonucleotide.

[0012] Optionally, the overlapping primer oligonucleotides are DNA oligonucleotides.

[0013] Optionally, the overlapping primer oligonucleotides are RNA oligonucleotides.

[0014] Optionally, the second sequence on the first single-stranded oligonucleotide is different from the second sequence on the second single-stranded oligonucleotide.

[0015] Advantageously, when the second sequence differs between the first and second single-stranded oligonucleotides, more diverse sequences can be incorporated into the extended oligonucleotide by complementary base pairing, resulting in two distinct fourth sequences being incorporated into the nucleic acid sequence.

[0016] Preferably, the first and third sequences are each at least 6 bases in length, preferably at least 8 bases in length.

[0017] Preferably, the second and fourth sequences are each at least 2 bases in length.

[0018] Optionally, at least one of the first and third sequences comprises at least one functional site.

[0019] Optionally, at least one functional site, in addition to being self-complementary, is a palindromic sequence (and may be considered a separate sequence from the first and third sequences, which are self-complementary palindromic sequences).

[0020] Optionally, at least one functional site is itself a self-complementary palindromic sequence.

[0021] Optionally, at least one functional site is a restriction enzyme site and / or a capping site.

[0022] Optionally, the at least one functional site may be selected from a restriction enzyme site, a capping site, a chemically reactive modification to a base, such as an alkyne, azide, or halo modification, a sequence-specific drug binding site, or an enzyme inhibition site.

[0023] With respect to functional moieties that are chemically reactive modifications to a base, at least one functional moiety is selected from an alkyne modification, an azide modification, and a halo modification.

[0024] For functional sites that are two or more modified bases, at least one functional site is selected from a restriction enzyme site, a capping site, a sequence-specific drug binding site, an enzyme inhibitor site, and the like.

[0025] Advantageously, by including a restriction site in one of the sequences, it provides the means for easily cutting nucleic acid sequence into shorter sequences.For example, sequence A and sequence C can code two desired oligonucleotides, and sequence B and sequence D can code two restriction enzyme binding sites.In this example, after generating long repeat sequences according to the method described herein, they can easily shorten long repeat sequences into desired oligonucleotide sequences by using relevant restriction enzymes.

[0026] Optionally, at least one of the first and third sequences comprises tandem repeats.

[0027] Optionally, both the first sequence and the third sequence comprise tandem repeats.

[0028] Optionally, the tandem repeat is a dinucleotide tandem repeat.

[0029] Advantageously, when the first and third sequences comprise dinucleotide tandem repeats, controlled incorporation of modified bases into the second and fourth sequences is possible.

[0030] Optionally, at least one of the second and fourth sequences comprises at least one modification selected from a modified nucleotide, an artificial base, or a loop structure.

[0031] Advantageously, modified nucleotides can be easily incorporated into nucleic acid sequences using the methods of the present invention. Modified nucleotides are used to alter the properties of nucleic acids, which are particularly desirable in fields such as nanotechnology, biomedical, and diagnostics.

[0032] Optionally, the modified nucleotide is an alkyne-, azide-, or phosphorothioate-modified nucleotide.

[0033] If necessary, modified nucleotides include 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP, 5-(octadiynyl)-dCTP, dye-labeled nucleotides, quencher-labeled nucleotides, endogenously fluorescent nucleotides, α-phosphate-modified nucleotides, γ-[(6-aminohexyl)-imido]-ATPαS, α,β non-hydrolyzable nucleotides, β-phosphate-modified nucleotides, β,γ non-hydrolyzable nucleotides, γ-phosphate-modified nucleotides, non-hydrolyzable dinucleotides, non-hydrolyzable dye-labeled nucleotides, biotin-labeled nucleotides, desthiobiotin-labeled nucleotides, digoxigenin-labeled nucleotides, DNP (dinitrophenol)-labeled nucleotides, photolabile group-labeled nucleotides, DBCO-labeled nucleotides, TCO-labeled nucleotides, vinyl-labeled nucleotides, free amino group (-NH2)-labeled nucleotides, redox dye-labeled nucleotides, halogen atom-labeled nucleotides, and water. The nucleotides may include silver labeled nucleotides, selenium labeled nucleotides, ferrocene labeled nucleotides, cap analogs and derivatives, puromycin analogs and derivatives, coenzyme A (CoA) analogs and derivatives, NAD analogs and derivatives, natural RNA nucleobase analogs and derivatives, cyclic dinucleotides, 3',5'-cyclic nucleotides, 2',3'-cyclic nucleotides, dinucleoside polyphosphates, 6-thiopurines, 7-deazapurines, 7-methylguanosine, substituted pyrimidines, 5-methylcytidine and related compounds, unmodified and modified ddNTPs, 2'-fluoro-2'-NTPs, 2'-O-methyl-NTPs, LNA-NTPs, cleavable base labeled dNTPs, N1-modified purines, N6-modified purines, 6-modified purines, 8-oxoguanosine, 2'-deoxyuridine, 3'-deoxynucleotides, nucleoside bisphosphates, aranucleotides, unmodified purines, modified dNTPs, 3'-O-azidomethyl-dNTPs.

[0034] Optionally, the modified nucleotide includes a linker that allows the modification to be attached to the nucleotide.

[0035] Advantageously, the use of a linker to attach the modification to the base prevents the modification from interfering with the base recognition site.

[0036] Optionally, the loop structure is a G-quadruplex region or a C-motif or an intercalation motif (i-motif) DNA.

[0037] Preferably, the method comprises: (iii) denaturing the duplex to provide first and second single-stranded polynucleotides; (iv) annealing the first and second single-stranded polynucleotides to provide an overhang region at the 5' end of at least one of the first or second single-stranded polynucleotides to allow formation of a polynucleotide duplex between the first single-stranded polynucleotide and the second single-stranded polynucleotide; (v) enzymatically extending both the first single-stranded polynucleotide and the second single-stranded polynucleotide to form a duplex; Further includes:

[0038] Preferably, steps (iii) to (v) are repeated to increase the length of the nucleic acid sequence.

[0039] Optionally, the overlapping primer oligonucleotides are immobilized on a surface.

[0040] Preferably, the overlapping primer oligonucleotides are immobilized to the surface by the 5' end of one of the first or second single-stranded polynucleotides.

[0041] Preferably, the surface comprises glass, silica, gold, graphene, graphene oxide, epoxy, plastic, metal, gel matrix, template exfoliated metal, or a composite thereof.

[0042] Optionally, the overlapping primer oligonucleotides are immobilized to a surface by covalent or non-covalent attachments.

[0043] Optionally, the overlapping primer oligonucleotides are immobilized to chemically modified regions of the surface.

[0044] Optionally, the overlapping primer oligonucleotides are immobilized to a surface by a linker.

[0045] Optionally, the linker comprises a silane linker molecule, a biotin-streptavidin complex, a thiol-Au linker, a Si-C covalent bond to silicon, a Si-O covalent bond to silicon, a Si-N covalent bond to silicon, a nanoparticle linker, or a dynamic covalent bond.

[0046] Preferably, the step of providing at least one overlapping primer oligonucleotide comprises obtaining a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, and hybridizing the first and second single-stranded oligonucleotide under suitable conditions.

[0047] According to a second aspect of the present invention, there is provided an overlapping primer oligonucleotide for extending the length of a repeat sequence of a nucleic acid, the overlapping primer oligonucleotide comprising two partially complementary single-stranded oligonucleotides, each single-stranded oligonucleotide comprising at least a first, second and third sequence, the third sequence being located at the 3' end of the first and second single-stranded oligonucleotides, the first and third sequences being palindromic self-complementary sequences, the second sequence being located between the first and third sequences, and the third sequence of the first single-stranded oligonucleotide hybridizing to the third sequence of the second single-stranded oligonucleotide to provide an overhang region at the 5' end of at least one of the first or second single-stranded oligonucleotides.

[0048] Optionally, the second sequence on the first single-stranded oligonucleotide is different from the second sequence on the second single-stranded oligonucleotide.

[0049] Optionally, the second sequence on the first single-stranded oligonucleotide is not complementary to the second sequence on the second single-stranded oligonucleotide.

[0050] Preferably, the first sequence and the third sequence are each at least 6 bases in length, preferably at least 8 bases in length.

[0051] Preferably, the second sequence is at least 2 bases in length.

[0052] Optionally, at least one of the first and third sequences comprises at least one functional site.

[0053] Optionally, the at least one functional site may be selected from a restriction enzyme site, a capping site.

[0054] Optionally, at least one of the first and third sequences comprises tandem repeats.

[0055] Optionally, both the first sequence and the third sequence comprise tandem repeats.

[0056] Optionally, the tandem repeat is a dinucleotide tandem repeat.

[0057] Optionally, the second sequence comprises at least one modification selected from a modified nucleotide, an artificial base, and a loop structure.

[0058] Optionally, the modified nucleotides are alkyne-, azide-, or phosphorothioate-modified nucleotides, and may include 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP, and 5-(octadiynyl)-dCTP.

[0059] Optionally, the modified nucleotide comprises a linker that allows the modification to be attached to the nucleotide.

[0060] Optionally, the loop structure is a G-quadruplex region.

[0061] Optionally, the overlapping primer oligonucleotides are DNA primer oligonucleotides.

[0062] Optionally, the overlapping primer oligonucleotides are RNA primer oligonucleotides.

[0063] Various further features and aspects of the present invention are defined in the claims.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0065] A "palindromic sequence" is a nucleic acid sequence in a double-stranded DNA or RNA molecule that is identical in reading in a particular direction (e.g., 5' to 3') on one strand to a sequence in the same direction (e.g., 5' to 3') on the complementary strand.

[0066] A "duplex" is a double-stranded nucleic acid sequence comprising two complementary sequences annealed to each other. A "partial duplex" is a double-stranded nucleic acid sequence in which a portion of one strand is complementary to the other strand and anneals to form a partial duplex, but the entire length of the strands is not complementary, resulting in a single-stranded polynucleotide tail at at least one end of the partial duplex.

[0067] The terms "hybridization," "binding," and "annealing" (or "hybridize," "bind," "anneal") in the context of nucleotide sequences are used interchangeably herein. The ability of two nucleotide sequences to hybridize to each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the proportion of matching complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the hybridization conditions can be and the more specific the binding of the two sequences. Increasing stringency is usually achieved by increasing the temperature, increasing the ratio of cosolvents, decreasing salt concentration, and other such methods known in the art.

[0068] The term "hybridization conditions" refers to the reagents and reaction conditions (e.g., temperature, time, etc.) used for hybridization. Generally, hybridization conditions can be stringent or moderate. Hybridization conditions used in the context of the methods described herein can be either moderate or stringent, since they allow for the formation of mismatched duplexes. Preferably, hybridization between the unit sequence of a first single-stranded oligonucleotide and a second single-stranded oligonucleotide forms a stable duplex at 65°C or below. Mismatched duplexes are preferably formed at temperatures up to 65°C, e.g., 55°C to 65°C, for 1 to 30 seconds as needed.

[0069] Moderate and stringent conditions are known to those skilled in the art and can be found in available references (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods are described in the references, and either can be used. A preferred example of stringent hybridization conditions is hybridization in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2× SSC, 0.1% (w / v) SDS at 50° C. Another example of stringent hybridization conditions is hybridization in 6× SSC at about 45° C., followed by one or more washes in 0.2× SSC, 0.1% (w / v) SDS at 55° C. A further example of stringent hybridization conditions is hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% (w / v) SDS at 60° C. Preferably, stringent hybridization conditions are hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% (w / v) SDS at 65° C. Particularly preferred stringency conditions (and the conditions to be used if the practitioner is unsure what conditions to apply to determine whether a molecule is within the hybridization limits of the invention) are 0.5 molar sodium phosphate, 7% (w / v) SDS at 65° C., followed by one or more washes in 0.2×SSC, 1% (w / v) SDS at 65° C.

[0070] The terms "extension" or "lengthening" in the context of a nucleotide sequence are used interchangeably herein. They refer to the extension of the 3' and / or 5' ends of a polynucleotide by the addition of nucleotides or bases. Chain extension in the context of the present invention is generally template dependent, i.e., the nucleotides added are determined by the sequence of the template nucleic acid to which the extension strand is hybridized.

[0071] The term "enzymatic extension" refers to the extension of a nucleic acid catalyzed by an enzyme, such as a polymerase enzyme. The polymerase enzyme is preferably template-dependent, such as Deep Vent® polymerase.

[0072] The term "extension conditions" refers to the reagents and reaction conditions (e.g., temperature, time, etc.) used. This describes the conditions for extension of a primer polynucleotide. In the present invention, contact between a mismatched duplex, a polymerase, and nucleotides is performed under extension conditions that allow polynucleotide extension in the 5' to 3' direction. Suitable extension conditions are known in the art. Preferably, extension is carried out at a temperature of about 65°C to 75°C, for 30 to 120 seconds as needed. Suitable conditions can be found, for example, in Whitfield CJ, Turley AT, Tuite EM, Connolly BA, Pike A R. Enzymatic Method for the Synthesis of Long DNA Sequences with Multiple Repeat Units. Angewandte Chemie International Edition 2015, 54(31), 8971-8974.

[0073] The term "complementary" in the context of nucleotide sequences refers to when one sequence can bind to another in an antiparallel manner, with the 3' end of one sequence binding to the 5' end of the other and each of the A, T(U), G, and C of one sequence aligning with the T(U), A, C, and G of the other sequence, respectively.

[0074] The term "amplification" as applied to nucleic acids refers to any method that results in the formation of one or more copies of a nucleic acid, where the amplification is preferably exponential. One such method for enzymatically amplifying a specific DNA sequence is known as the polymerase chain reaction (PCR), as described in Saiki et al. (1986, Science 230:1350-1354).

[0075] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to any nucleic acid, including DNA, RNA, cDNA, DNA-RNA, peptide nucleic acid (PNA), hybrids, or mixtures thereof.

[0076] The terms "nucleic acid," "polynucleotide," and "nucleotide" also specifically include nucleic acids composed of synthetic bases (i.e., bases other than the five biological bases, adenine, guanine, thymine, cytosine, and uracil), modified bases, or any combination of biological, synthetic, and modified bases.

[0077] The term "SS oligonucleotide" refers to a "single-stranded oligonucleotide."

[0078] The polynucleotides of the invention may be derived from a human or non-human mammal, or from other organisms, may be derived from any recombinant source, may be synthesized in vitro, or may be synthesized by chemical synthesis.

[0079] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which like parts have corresponding reference numerals and in which: [Brief explanation of the drawings]

[0080] [Figure 1] 1 is a schematic diagram of a method according to the present invention; [Figure 2] 1 shows the UV absorption of PCR products after increasing the number of PCR cycles using the double-stranded primer oligonucleotide of Example 1. [Figure 3A] 1 is a visualization of gel electrophoresis of PCR products after 3, 4, 10, and 20 PCR cycles using the double-stranded primer oligonucleotide of Example 1. [Figure 3B] Fluorescence intensity of the gel electrophoresis shown in FIG. 3A is shown. [Figure 4]1 shows the UV absorption of PCR products after increasing the number of PCR cycles using the double-stranded primer oligonucleotide of Example 2. [Figure 5A] 1 is a visualization of gel electrophoresis of PCR products after 3, 4, 10, and 20 PCR cycles using the double-stranded primer oligonucleotide of Example 2. [Figure 5B] Fluorescence intensity of the gel electrophoresis shown in FIG. 5A is shown. [Figure 6] 1 shows the double-stranded primer oligonucleotide and extended polynucleotide product of Example 2. [Figure 7] 1 shows the UV absorption of PCR products after increasing the number of PCR cycles using the double-stranded primer oligonucleotide of Example 3. [Figure 8A] 1 is a visualization of gel electrophoresis of PCR products after 3, 4, 10, and 20 PCR cycles using the double-stranded primer oligonucleotide of Example 3. [Figure 8B] Fluorescence intensity of the gel electrophoresis shown in FIG. 8A is shown. [Figure 9] The UV / vis spectrum of the purified extended DNA is shown. [Figure 10A] Visualization of gel electrophoresis of extended DNA against Gene Ruler 1kb plus DNA ladder is shown, and the fluorescence intensity of the gel electrophoresis was measured and analyzed using Image-J software. [Figure 10B] FIG. 10 is a visualization of gel electrophoresis of digestion products against a low-range ladder, and the fluorescence intensity of the gel electrophoresis was measured and analyzed using Image-J software. DETAILED DESCRIPTION OF THE INVENTION

[0081] Figure 1 shows a schematic diagram of the method of the present invention. An initial overlap primer oligonucleotide 100 comprises two single-stranded (SS) oligonucleotides 100a and 100b. Each of the SS oligonucleotides 100a and 100b comprises the sequences A, B, and C. The first SS oligonucleotide 100a comprises, from 5' to 3', the sequences A, B, and C. The second SS oligonucleotide 100b comprises, from 3' to 5', the sequences C, B, and A.

[0082] In this embodiment, sequence A is a palindromic self-complementary sequence, sequence C is a palindromic self-complementary sequence, and all sequences are identical in each SS oligonucleotide. Sequence B is complementary to sequence D, the fourth sequence, which is incorporated into the SS oligonucleotide by complementary base pairing as described below. Sequences A and C are at least 6 bases long, preferably 8 bases long. Sequences B and D are at least 2 bases long.

[0083] It will be apparent to those skilled in the art that the nucleotide sequences of sequences B and D may differ between the first and second SS oligonucleotides, provided that sequence B of the first SS oligonucleotide is complementary to sequence D of the second SS oligonucleotide and sequence B of the second SS oligonucleotide is complementary to sequence D of the first SS oligonucleotide.

[0084] The overlap primer oligonucleotide 100 is formed by hybridization of sequence C of the first SS oligonucleotide 100a with sequence C of the second SS oligonucleotide 100b. This hybridization results in the formation of a partial duplex having an overhang region at the 5' end of each of the SS oligonucleotide sequences 100a and 100b. The overhang region of each SS oligonucleotide comprises both sequences A and B.

[0085] The overlapping primer oligonucleotides 100 are extended 101 in the presence of an appropriate polymerase and nucleotides under extension conditions. Any suitable polymerase, such as Deep Vent DNA polymerase (New England Biolabs), can be used. Several known thermostable 5' to 3' polymerases are available and can be used in the methods described herein. The polymerase preferably has thermostability and high stability, such that its activity is substantially retained during the long incubation times required for the extension reaction. The polymerase preferably has high processivity. The polymerase preferably does not exhibit nonspecific nuclease activity. The polymerase preferably has good fidelity, but can accept a variety of nucleotide analogs as both templates and substrates. High-fidelity polymerases with high-fidelity proofreading activity are not particularly suitable. Preferably, the polymerase lacks 3' to 5' exonuclease activity [3' to 5' exo(-)], resulting in low fidelity due to the lack of proofreading function. Those skilled in the art can determine whether a particular polymerase has the required properties defined above. Exemplary polymerases include, but are not limited to, Thermococcus gorgonarius family B polymerase (Tgo-Pol) enzyme variant, Z3 (Tgo-Pol Z3) exo(-) [Jozwiakowski et al., 2011 Chembiochem 12: 35-37], Deep Vent exo(-) (New England Biolabs), Vent exo(-) (New England Biolabs), Pfu exo(-) (Agilent Technologies), and Taq polymerase (from many suppliers).

[0086] Extension of the overlapping primer oligonucleotides can be carried out using a PCR reaction, which extends the SS oligonucleotides 100a and 100b in the overhang region to form a stable duplex 107. In this step, a fourth sequence (D) is incorporated into the SS oligonucleotides 100a and 100b. Sequence D is the complementary sequence of sequence B.

[0087] To further extend the sequence, the stable duplex 107 is denatured 102 and reannealed under appropriate conditions to form a second partial duplex 108. The second partial duplex 108 is formed by hybridization of sequence A of the first SS oligonucleotide 100a with sequence A of the second SS oligonucleotide 100b. This hybridization results in the generation of a mismatched duplex having an overhang region at the 5' end of each of the SS oligonucleotide sequences 100a and 100b. The overhang region of each SS oligonucleotide comprises sequences B, C, and D once and sequence A twice.

[0088] In some embodiments, the sequences are designed to have different melting temperatures (Tms), which allows for control of the amount of mismatch, but when the Tms are similar, 50% are expected to form mismatched duplexes that can be extended.

[0089] The second partial duplex 108 is extended 103 in the presence of an appropriate polymerase and nucleotides under extension conditions, thereby extending the SS oligonucleotides 100a, 100b in the overhang region to form a second stable duplex 109. Furthermore, sequences A, B, C, and D are incorporated into each SS oligonucleotide 100a, 100b, with sequences B, C, and D appearing twice and sequence A appearing three times in each SS oligonucleotide.

[0090] To further extend the SS oligonucleotides 100a and 100b, the second stable duplex 109 is denatured 104. Further extension can occur under maximum overlap conditions 105 or minimum overlap conditions 106. Under maximum overlap conditions 105, the SS oligonucleotides 105a are annealed so that there is a maximum amount of overlap between the first and second SS oligonucleotides 100a and 100b. Under maximum overlap conditions 105, a third partial duplex 110 is formed by hybridization of the sequences A, B, C, D, and A of the first SS oligonucleotide 100a with the sequences A, D, C, B, and A of the second SS oligonucleotide 100b. This hybridization results in a third mismatched duplex 110 having overhang regions at the 5' ends of each of the SS oligonucleotide sequences 100a and 100b. The overhang regions of each SS oligonucleotide comprise the sequences A, B, C, and D.

[0091] By changing the temperature of the denaturation and / or annealing step, the reaction can be directed to the minimum overlap condition. For example, by increasing the denaturation and annealing temperature, the reaction can be directed to the minimum overlap condition. It is also possible that after several cycles of extension reaction, the reaction can be directed to the minimum overlap condition by increasing only the annealing temperature. Directing the reaction to the minimum overlap condition is advantageous because it will produce longer products on average.

[0092] The third partial duplex 110 is extended 105b in the presence of an appropriate polymerase and nucleotides under extension conditions, thereby extending the SS oligonucleotides 100a, 100b at the overhang regions to form a third stable duplex 111. Furthermore, sequences A, B, C, and D are incorporated into each SS oligonucleotide 100a, 100b, such that each SS oligonucleotide contains sequences B, C, and D three times and sequence A four times.

[0093] Under minimum overlap conditions 106, the SS oligonucleotides are annealed 106a so that there is a minimum amount of overlap between the first SS oligonucleotide 100a and the second SS oligonucleotide 100b. Under minimum overlap conditions 106, a fourth partial duplex 112 is formed by hybridization of sequence A of the first SS oligonucleotide 100a with sequence A of the second SS oligonucleotide 100b. This hybridization produces a fourth partial duplex 112 having overhang regions at the 5' ends of each of the SS oligonucleotide sequences 100a, 100b. The overhang regions of each SS oligonucleotide comprise sequences A, B, C, and D twice.

[0094] The fourth partial duplex 112 is extended 106b in the presence of an appropriate polymerase and nucleotides under extension conditions, thereby extending the SS oligonucleotides 100a, 100b in the overhang regions to form a fourth stable duplex 113. Two more of sequences A, B, C, and D are incorporated into each of the SS oligonucleotides 100a, 100b, such that each SS oligonucleotide contains four occurrences of sequences B, C, and D, and five occurrences of sequence A.

[0095] The denaturation step 104, the annealing steps 105a, 106a, and the extension steps 105b, 106b may be repeated any number of times to continue extending the sequence. [Example]

[0096] Example 1 5’ -(AT)5C2(TA)5- 3’ / 3’ -(AT)5C2(TA)5- 5’ ) In one embodiment of the present invention, the initial overlap primer oligonucleotides comprised a first SS oligonucleotide and a second SS oligonucleotide. All custom oligonucleotides were purchased from Eurofins (Ebersberg, Germany). The first and second SS oligonucleotides comprised identical sequences A, B, and C, respectively. The nucleotide sequences of sequences A, B, and C are shown in Table 1.

[0097] [Table 1]

[0098] The first SS oligonucleotide is 5’ -ABC- 3’ (SEQ ID 5), and the second SS oligonucleotide has a sequence in the order: 3’ -CBA- 5’ (SEQ ID 6).

[0099] Sequences A and C are palindromic self-complementary sequences, so that the first and second SS oligonucleotides hybridize to form an overlapping primer oligonucleotide, which is duplex with a 5' overhang region containing sequences A and B, as shown at 100 in Figure 1. The overlapping primer oligonucleotide is 22 base pairs in length. Sequence D is complementary to sequence B and is incorporated into the oligonucleotide during enzymatic extension. The nucleotide sequence of sequence D is shown in Table 1 above.

[0100] In this example, sequences A and C comprise a dinucleotide repeat sequence of bases A and T. While sequences A and C are palindromic self-complementary sequences and contain only two of the four DNA nucleotide bases (A and T in this example), modified bases can be incorporated into sequences B and D in a controlled manner. Advantageously, this allows the user greater control over where the modified bases are incorporated, and therefore greater control over the composition and properties of the extended polynucleotide product. Those skilled in the art will understand that this is only one example of the invention, and that in other examples, sequences A and C can contain sequences other than dinucleotide repeat sequences. Similarly, those skilled in the art will understand that in other examples, sequences B and D can contain sequences other than mononucleotide repeat sequences.

[0101] Figure 2 shows the UV absorption of PCR products after increasing the number of PCR cycles. The increase in UV light absorption at a wavelength of 260 nm indicates an increase in DNA concentration. Figure 2 shows that DNA concentration increases with increasing PCR cycles, indicating that new DNA is generated with increasing PCR cycles.

[0102] Figure 3A shows gel electrophoresis visualization of PCR products after 3, 4, 10, and 20 PCR cycles. The fluorescence intensity of the gel electrophoresis was measured and analyzed using Image-J software, and the results are shown in Figure 3B. As can be seen in Figure 3B, after 20 PCR cycles, DNA products with lengths of 1,000 to 5,000 base pairs can be detected.

[0103] Example 2 5’ -(AT)5C4(TA)5- 3’ / 3’ -(AT)5C4(TA)5- 5’ ) In another embodiment of the present invention, the initial overlap primer oligonucleotides comprised first and second SS oligonucleotides, each comprising identical sequences A, B, and C. The nucleotide sequences of sequences A, B, and C are shown in Table 2.

[0104] [Table 2]

[0105] The first SS oligonucleotide is 5’ -ABC- 3’ (SEQ ID 11), and the second SS oligonucleotide has a sequence in the order: 3’ -CBA- 5’ (SEQ ID 12).

[0106] Sequences A and C are palindromic self-complementary sequences, such that the first and second SS oligonucleotides hybridize to form a duplex with a 5' overhang containing sequences A and B, as shown at 100 in Figure 1. Sequence D is complementary to sequence B and is incorporated into the oligonucleotide during enzymatic extension. The nucleotide sequence of sequence D is shown in Table 2 above.

[0107] UV absorbance was measured as described in Example 1. Figure 4 shows the UV absorbance data. An increase in absorbance of UV light at a wavelength of 260 nm indicates an increase in DNA concentration. As can be seen from Figure 4, the DNA concentration in the PCR product increases with increasing PCR cycle number, indicating that new DNA is generated with increasing PCR cycle number.

[0108] Figure 5A shows gel electrophoresis visualization of PCR products after 3, 4, 10, and 20 PCR cycles. The fluorescence intensity of the gel electrophoresis was measured and analyzed using Image-J software, and the results are shown in Figure 5B. As can be seen in Figure 5B, after 20 PCR cycles, DNA products with lengths of 1,000 to 5,000 base pairs can be detected.

[0109] Example 3 5’ -(AT)5C2(TA)5- 3’ / 3’ -(AT)5C8(TA)5- 5’ ) In another embodiment of the invention, the initial overlap primer oligonucleotides comprised first and second SS oligonucleotides.

[0110] The first SS oligonucleotide had sequence A, sequence B, and sequence C. The second SS oligonucleotide had sequence A, sequence B', and sequence C. The nucleotide sequences of sequences A, B, C, and B' are shown in Table 3.

[0111] [Table 3]

[0112] The first SS oligonucleotide is 5’ -ABC- 3’ (SEQ ID 19), and the second SS oligonucleotide has a sequence in the order: 3’ -CBA- 5’ (SEQ ID 20).

[0113] As in the previous example, sequence A is a palindromic self-complementary sequence, and sequence C is a palindromic self-complementary sequence. As shown in Figure 6, duplex 200 is formed by hybridization of sequence C of first SS oligonucleotide 200a and second SS oligonucleotide 200b. Duplex 200 comprises 5' overhangs of sequences A and B of first SS oligonucleotide 200a and sequences B' and A of second SS oligonucleotide 200b.

[0114] During enzymatic extension, complementary base pairing occurs, extending the first and second SS oligonucleotides at their 3' ends. The first SS oligonucleotide is extended to include sequences D' and A in the extended first SS oligonucleotide 201a. Sequence D' is complementary to sequence B'. The second SS oligonucleotide is extended to include sequences A and D in the second extended SS oligonucleotide 201b. Sequence D is complementary to sequence B. The nucleotide sequences of sequences D and D' are set forth in Table 3 above.

[0115] UV absorbance was measured as described in Example 1. Figure 7 shows the UV absorbance data. An increase in absorbance of UV light at a wavelength of 260 nm indicates an increase in DNA concentration. As can be seen from Figure 7, the DNA concentration in the PCR product increases with increasing PCR cycle number, indicating that new DNA is generated with increasing PCR cycle number.

[0116] Figure 8A shows gel electrophoresis visualization of PCR products after 3, 4, 10, and 20 PCR cycles. The fluorescence intensity of the gel electrophoresis was measured and analyzed using Image-J software, and the results are shown in Figure 8B. As can be seen in Figure 8B, after 20 PCR cycles, DNA products with lengths of 1,000 to 5,000 base pairs can be detected.

[0117] (material and method) (Preparation of overlapping primer oligonucleotides) Overlapping primer oligonucleotides (sequences shown in Tables 1, 2, and 3) were prepared by adding the first and second SS oligonucleotides (10 μL of each SS oligonucleotide (100 μM)) to 480 μL of 1X HEPES and potassium acetate DNA annealing buffer in an Eppendorf flask. The HEPES and potassium acetate buffer was made from 10 mM HEPES, 100 mM KCl, and 1 mM EDTA. The solution was vortexed for a few seconds to mix thoroughly, heated at 95°C for 10 minutes, and then slowly cooled to room temperature (approximately 25°C). The overlapping primer oligonucleotides were stored at -20°C in a freezer when not in use.

[0118] (DNA elongation) Nanopure-HO (60 μL) was added to 10 μL each of dATP, dTTP, dCTP, and dGTP (all 100 mM) to prepare 100 μL of 10 mM dNTP mixture. For each example, primer-specific dNTP mixtures were prepared by varying the amount of dNTP added depending on the ratio of bases present in the overlapping primer oligonucleotides. For example, the overlapping primer oligonucleotides ((AT)C2(TA)5 / (AT)C2(TA)5) used in Example 1 had an A:T:C:G ratio of 5:5:1:1, and the dNTPs were mixed in the same A:T:C:G ratio. For example, dATP (16.6 μL, 100 mM), dTTP (16.6 μL, 100 mM), dCTP (3.4 μL, 100 mM), and dGTP (3.4 μL, 100 mM) were added to nanopure-HO (60 μL) to make the dNTP mixture used in Example 1.

[0119] (heating and cooling cycle) The extension reaction mixture was prepared in a thin-walled 200 μL Eppendorf tube by adding nanopure HO (36 μL) to ThermoPol buffer (5 μL, 10X), overlapping primer oligonucleotides (5 μL, 2 μM), primer-specific dNTP mix (2.5 μL, 10 mM), and MgSO (1 μL, 100 mM). The mixture was then vortexed for a few seconds before adding Deep Vent DNA polymerase enzyme (0.5 μL, 1 U). At 75°C, 10 nmol of dNTPs can be incorporated per unit of Deep Vent DNA polymerase enzyme. To avoid disrupting the polymerase enzyme, the mixture was gently mixed with a pipette tip instead of using a vortex. After 10 cycles, the primer-specific dNTP mix (2.5 μL, 10 mM) was added along with additional polymerase enzyme (0.5 μL, 1 U). The Eppendorf was then placed back into the thermocycler and run for 10 more cycles (20 cycles total) using the same settings. Reagents and products were stored at -20°C when not in use.

[0120] Each cycle is 1) denaturing at 95°C for 30 seconds; 2) annealing at 55°C for 30 seconds; 3) 72°C for 120 seconds; It consists of:

[0121] After 10 cycles, the thermocycler cooled the mixture to 4°C and held the mixture at this temperature for the required period.

[0122] In each example above, a different number of cycles was performed: for 20 cycles, the procedure above was followed; for 10 cycles, the first half of the procedure above was performed (i.e., the experiment was stopped after the first 10 cycles, eliminating the need for a second addition of dNTP mix and polymerase enzyme).

[0123] (Purification of elongated DNA products) For purification, the Monarch® PCR and DNA Cleanup Kit was used. DNA binding buffer (100 μL) was added to the extended DNA solution and mixed with a pipette before transferring the entire sample to a thick-walled tube and column. The column was centrifuged at 13,000 RPM for 1 minute, and the waste buffer was then disposed of as aqueous waste. DNA wash buffer (200 μL) was added to the column, which was then centrifuged at 13,000 RPM for 1 minute. This step was repeated, with all washes disposed of as aqueous waste. The column was centrifuged again at 13,000 RPM for 1 minute, ensuring all excess liquid was removed and the column was completely dry. The column (with the DNA attached to the silica) was placed in a 1.5 mL Eppendorf tube, which was heated to 65°C in a heating block. This heating step was intended to increase yield. Elution buffer (20 μL) was added to the column in the heating block and held at 65°C for 5 minutes. The sample was finally centrifuged at 13,000 RPM for 1 minute. The purified extended DNA product was collected in a 1.5 mL Eppendorf tube. All binding / washing / elution buffers were stored at room temperature.

[0124] (UV / Vis spectroscopy) UV / Vis spectra, overlap primer oligonucleotide concentrations, and purity ratios were recorded using a Thermo Scientific™ NanoDrop™ One microvolume UV / Vis spectrophotometer, which uses surface tension to hold a small sample volume between two pedestals, allowing for analysis of 1 μL samples. Most sample and blank measurements were recorded using the dsDNA setting. Blanks were obtained using Monarch® DNA Elution Buffer before each DNA sample measurement. Between measurements, the pedestals were cleaned with lint-free wipes to minimize contamination between samples. The presence of DNA was confirmed by an absorption band at 260 nm.

[0125] (Agarose gel electrophoresis) Gels were electrophoresed at 100 V (400 mA) for 60-90 minutes. All gels were imaged using UviProMW1 software and a Uvitec Chemiluminescence fluorescent imaging box. All gels were analyzed using ImageJ software, and data were converted into graphs using Microsoft Excel.

[0126] Example 4: Inclusion of restriction sites In another embodiment of the present invention, a restriction site is included in the initial overlap primer oligonucleotide. After extension, the nucleic acid can be cleaved using the restriction site to provide two oligonucleotides of different lengths. The nucleotide sequences of sequences A, B, C, D, and E are shown in Table 4.

[0127] [Table 4]

[0128] In this example, A is complementary to E, C is complementary to D, B is a self-complementary restriction site that splits into two parts after digestion with a restriction enzyme, adding three bases to each end of the oligomer.

[0129] After extension, the product is a long double-stranded 5'-[ABCB] n -3' / 3'-[EBDB] n It becomes -5'.

[0130] Restriction enzyme digestion yields two double-stranded oligomers of 18 and 13 bases derived from the A / E and C / D complementary sequences, respectively.

[0131] JPEG0007815482000005.jpg20127

[0132] JPEG0007815482000006.jpg29166

[0133] Overlapping primer oligonucleotides (sequences shown below) were prepared by adding the first and second SS oligonucleotides (10 μL of each SS oligonucleotide (100 μM)) to 480 μL of 1X HEPES and potassium acetate DNA annealing buffer in an Eppendorf. The HEPES and potassium acetate buffer was made from 10 mM HEPES, 100 mM KCl, and 1 mM EDTA. The solution was vortexed for a few seconds to mix thoroughly, heated at 95°C for 10 minutes, and then slowly cooled to room temperature (approximately 25°C). The overlapping primer oligonucleotides were stored at -20°C in a freezer when not in use.

[0134] JPEG0007815482000007.jpg22166

[0135] Nanopure-H2O (60 μL) was added to 10 μL each of dATP, dTTP, dCTP, and dGTP (all 100 mM) to make 100 μL of dNTP mixture at a concentration of 10 mM.

[0136] The extension reaction mixture was prepared in a thin-walled 200 μL Eppendorf tube by adding nanopure HO (36 μL) to ThermoPol buffer (5 μL, 10X), overlapping primer oligonucleotides (5 μL, 2 μM), primer-specific dNTP mix (2.5 μL, 10 mM), and MgSO (1 μL, 100 mM). The mixture was then vortexed for a few seconds before adding Deep Vent DNA polymerase enzyme (0.5 μL, 1 U). At 75°C, 10 nmol of dNTPs can be incorporated per unit of Deep Vent (exo-) DNA polymerase enzyme. To avoid disrupting the polymerase enzyme, the mixture was gently mixed with a pipette tip instead of using a vortex. After 10 cycles, the primer-specific dNTP mix (2.5 μL, 10 mM) was added along with additional polymerase enzyme (0.5 μL, 1 U). The Eppendorf was then placed back into the thermocycler and run for 10 more cycles (20 cycles total) using the same settings. Reagents and products were stored at -20°C when not in use.

[0137] Each cycle is 1) denaturing at 95°C for 30 seconds; 2) annealing at 55°C for 30 seconds; 3) 72°C for 120 seconds; It consists of:

[0138] After 20 cycles, the thermocycler cooled the mixture to 4°C and held the mixture at this temperature for the required period.

[0139] For purification, the Monarch® PCR and DNA Cleanup Kit was used. DNA binding buffer (100 μL) was added to the extended DNA solution and mixed with a pipette before transferring the entire sample to a thick-walled tube and column. The column was centrifuged at 13,000 RPM for 1 minute, and the waste buffer was then disposed of as aqueous waste. DNA wash buffer (200 μL) was added to the column, which was then centrifuged at 13,000 RPM for 1 minute. This step was repeated, with all washes disposed of as aqueous waste. The column was centrifuged again at 13,000 RPM for 1 minute, ensuring all excess liquid was removed and the column was completely dry. The column (with the DNA attached to the silica) was placed in a 1.5 mL Eppendorf tube, which was heated to 60°C in a heating block. This heating step was intended to increase yield. Elution buffer (20 μL) was added to the column in the heating block, which was then held at 65°C for 5 minutes. The sample was finally centrifuged at 13,000 RPM for 1 minute. The purified extended DNA product was collected in a 1.5 mL Eppendorf tube. All binding / washing / elution buffers were stored at room temperature.

[0140] (UV / Vis spectroscopy) UV / Vis spectra, overlap primer oligonucleotide concentrations, and purity ratios, as shown in Figure 9, were recorded using a Thermo Scientific™ NanoDrop™ One microvolume UV / Vis spectrophotometer, which uses surface tension to hold a small sample volume between two pedestals, allowing for analysis of 1 μL samples. Most sample and blank measurements were recorded using the dsDNA setting. Blanks were obtained using Monarch® DNA Elution Buffer before each DNA sample measurement. Between measurements, the pedestals were cleaned with lint-free wipes to minimize contamination between samples. The presence of DNA was confirmed by an absorption band at 260 nm.

[0141] (Agarose gel electrophoresis) Gels were electrophoresed at 100 V (400 mA) for 60-90 minutes. All gels were imaged using UviProMW1 software and a Uvitec Chemiluminescence fluorescent imaging box. All gels were analyzed using ImageJ software, and data were converted into graphs using Microsoft Excel.

[0142] (digestion) A reaction mixture was made using the extended dsDNA product, rCutSmart buffer, and EcoRV HF at the concentrations and volumes listed in Table 5. The reaction mixture was heated at 37°C for 3 hours, after which purple loading dye was added to inactivate the enzyme.

[0143] [Table 5]

[0144] A 4% MetaPhor agarose gel was used to run the digestion products alongside a low range ladder, and the gel electrophoresis was analyzed using Image-J as shown in Figure 10 .

[0145] Figure 10(A) shows the visualization of gel electrophoresis of extended DNA against the Gene Ruler 1 kb plus DNA ladder. The fluorescence intensity of the gel electrophoresis was measured and analyzed using Image-J software, and the results show extension products of 75 to 300 base pairs.

[0146] Figure 10(B) shows the gel electrophoresis visualization of the digestion products against the low-range ladder. The fluorescence intensity of the gel electrophoresis was measured and analyzed using Image-J software. As can be seen above, after digestion, DNA bands of 31 bases (when excised at each restriction site), 18 bases, and 13 bases are detected, corresponding to the two products (A / E and C / D) expected after digestion on either side of the restriction site.

[0147] All features disclosed in this specification (including the accompanying claims, abstract, and drawings), and / or all steps of the disclosed methods or processes, may be combined in any combination, except combinations in which at least some features and / or steps are mutually exclusive. Each feature disclosed in this specification (including the accompanying claims, abstract, and drawings) may be replaced by an alternative feature serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each disclosed feature is merely an example of a generic series of equivalent or similar features. The present invention is not limited to the description of the foregoing embodiments. The present invention extends to any novel or novel combination of features disclosed in this specification (including the accompanying claims, abstract, and drawings), or any novel or novel combination of steps of the disclosed methods or processes.

[0148] With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art may convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0149] In general, those skilled in the art will understand that the terms used herein, and particularly in the appended claims, are generally intended as "open" terms (e.g., the terms "comprises" or "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.). Furthermore, those skilled in the art will understand that if a specific number of introduced claim recitations is intended, such intention will be expressly recited in the claim; in the absence of such recitation, no such intention exists. For example, to aid in understanding, the following appended claims may use the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" limits a particular claim that includes that introduced claim recitation to embodiments including only one such recitation. This is also true when the same claim includes the introductory phrase "one or more" or "at least one" and the indefinite article "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. Moreover, even when a specific number of introduced claims is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the mere recitation of "two recitations," without other modifiers, means at least two recitations, or more than two recitations).

[0150] It will be understood that various embodiments of the present disclosure have been described herein for purposes of illustration and that various changes may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope being indicated by the appended claims.

Claims

1. 1. A method for extending the length of a nucleic acid, comprising: (i) providing at least one overlapping primer oligonucleotide, the overlapping primer oligonucleotide comprising a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, each of the first single-stranded oligonucleotide and the second single-stranded oligonucleotide comprising at least a first, a second, and a third sequence, the third sequence being located at a 3' end of each single-stranded oligonucleotide, the second sequence being located between the first sequence and the third sequence, and the first sequence and the third sequence of the first single-stranded oligonucleotide and the second single-stranded oligonucleotide being self-complementary palindromic sequences; the first and third sequences of the first single-stranded oligonucleotide and the second single-stranded oligonucleotide are each at least 10 bases in length; the third sequence of the first single-stranded oligonucleotide hybridizes to the third sequence of the second single-stranded oligonucleotide to provide an overhang region at a 5' end of at least one of the first or second single-stranded oligonucleotide; the second sequence on the first single-stranded oligonucleotide is different from the second sequence on the second single-stranded oligonucleotide; Steps and (ii) enzymatically extending both the first single-stranded oligonucleotide and the second single-stranded oligonucleotide to form a duplex; A method for extending the length of a nucleic acid, comprising:

2. 2. The method of claim 1, wherein a fourth sequence is incorporated into the first and second single-stranded oligonucleotides during step (ii), the fourth sequence on the first single-stranded oligonucleotide being complementary to the second sequence on the second single-stranded oligonucleotide, and the fourth sequence on the second single-stranded oligonucleotide being complementary to the second sequence on the first single-stranded oligonucleotide.

3. (iii) denaturing the duplex to provide first and second single-stranded polynucleotides; (iv) annealing the first and second single-stranded polynucleotides to provide an overhang region at the 5' end of at least one of the first or second single-stranded polynucleotides, thereby allowing the formation of a polynucleotide duplex between the first single-stranded polynucleotide and the second single-stranded polynucleotide; (v) enzymatically extending both the first single-stranded polynucleotide and the second single-stranded polynucleotide to form a duplex; 3. The method for extending the length of a nucleic acid according to claim 1 or 2, further comprising:

4. 4. The method of claim 3, wherein steps (iii) through (v) are repeated to increase the length of the nucleic acid sequence.

5. 3. The method for extending the length of a nucleic acid according to claim 1 or 2, wherein the overlapping primer oligonucleotides are immobilized on a surface, preferably by the 5' end of one of the first or second single-stranded polynucleotides, and optionally the surface comprises glass, silica, gold, graphene, graphene oxide, epoxy, plastic, metal, a gel matrix, template-stripped metal, or a composite thereof.

6. 6. The method for extending the length of a nucleic acid according to claim 5, wherein the overlapping primer oligonucleotides are immobilized on the surface by a covalent or non-covalent bond, and / or the overlapping primer oligonucleotides are immobilized on the surface by a linker, preferably comprising a silane linker molecule, a biotin-streptavidin complex, a thiol-Au linker, a Si-C covalent bond to silicon, a Si-O covalent bond to silicon, a Si-N covalent bond to silicon, a nanoparticle linker, or a dynamic covalent bond, and / or the overlapping primer oligonucleotides are immobilized on a chemically modified region of the surface.

7. 1. An overlapping primer oligonucleotide for extending the length of a repeat sequence of a nucleic acid, said overlapping primer oligonucleotide comprising two partially complementary single-stranded oligonucleotides, each single-stranded oligonucleotide comprising at least a first, second and third sequence, said third sequence being located at the 3′ end of said first and second single-stranded oligonucleotides; the first sequence and the third sequence are palindromic self-complementary sequences, the second sequence is located between the first sequence and the third sequence, and the third sequence of the first single-stranded oligonucleotide hybridizes to the third sequence of the second single-stranded oligonucleotide to provide an overhang region at a 5' end of at least one of the first or second single-stranded oligonucleotide; the first and third sequences of the first single-stranded oligonucleotide and the second single-stranded oligonucleotide are each at least 10 bases in length; overlapping primer oligonucleotides for extending the length of a repeat sequence of a nucleic acid, wherein the second sequence on the first single-stranded oligonucleotide is different from the second sequence on the second single-stranded oligonucleotide and / or the second sequence on the first single-stranded oligonucleotide is not complementary to the second sequence on the second single-stranded oligonucleotide.

8. 3. The method for extending the length of a nucleic acid according to claim 1, wherein the second sequence is at least 2 bases in length.

9. 3. The method for extending the length of a nucleic acid according to claim 1 or 2, wherein at least one of the first and third sequences comprises at least one functional site, and the at least one functional site may be selected from a restriction enzyme site, a capping site, a sequence-specific drug binding site, and an enzyme inhibition site.

10. 3. The method of claim 1 or 2, wherein at least one of the first and third sequences comprises a tandem repeat, and optionally the tandem repeat is a dinucleotide tandem repeat.

11. The method for extending the length of a nucleic acid according to claim 1 or 2, wherein the second sequence comprises at least one modification selected from a modified nucleotide, an artificial base, and a loop structure.

12. 12. The method of claim 11, wherein the modified nucleotides are alkyne, azide, or phosphorothioate modified nucleotides, which may optionally include 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP, 5-(octadiynyl)-dCTP, and / or the modified nucleotides include a linker that allows the modification to be attached to the nucleotide.

13. 12. The method for extending the length of a nucleic acid according to claim 11, wherein the loop structure is a G-quadruplex region or a C-motif or an intercalation motif DNA.

14. 3. The method for extending the length of a nucleic acid according to claim 1, wherein the overlapping primer oligonucleotide is a DNA primer oligonucleotide or an RNA primer oligonucleotide.

15. 8. The overlapping primer oligonucleotide of claim 7, wherein at least one of the first and third sequences comprises a tandem repeat, and optionally the tandem repeat is a dinucleotide tandem repeat.

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