Nucleic Acids 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 Art

[0002] Nucleic acid synthesis is the process of forming nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by bonding nucleotides. Nucleic acids can be synthesized using various techniques such as phosphoramidite synthesis, slippage reactions, loop-mediated amplification (LAMP), primer-template extension, and thermal cycle amplification.

[0003] The synthesis of nucleic acids containing repetitive sequences is difficult with existing nucleic acid synthesis methods, especially for the synthesis of long sequences (over 200 base pairs (bp)). Usually, the challenges in synthesizing longer nucleic acid sequences are due to the decreased yield and quality when generating longer sequences. One of the most commonly used methods for nucleic acid synthesis is column-based oligonucleotide synthesis, where bases are sequentially added to a growing oligonucleotide sequence on a solid support using phosphoramidite synthesis chemistry. In this method, a four-step process of deblocking (detritylation), coupling, capping, and oxidation is utilized, and this is repeated to construct the oligonucleotide sequence. Once the synthesis is complete, the oligonucleotide is chemically cleaved from the solid support. This process forms the basis of many commercial gene synthesis systems as it is particularly suitable for automation. The main drawback of column-based oligonucleotide synthesis is that the yield and quality of the oligonucleotide product decrease with the length of the sequence. This is because during synthesis, pseudodepurination occurs, especially with adenosine. These pseudosites promote cleavage of the oligonucleotide backbone, resulting in a decreased yield. This decrease in yield and quality means that this method is not suitable for generating oligonucleotides longer than 200 bases in length. Phosphoramidite synthesis is also inefficient in generating sequences with a high GC content, hairpin structures, and / or highly repetitive sequences.

[0004] Alternatively, enzyme technologies can be used for nucleic acid synthesis. Usually, these methods have fewer restrictions regarding the length of the sequence compared to phosphoramidite synthesis. In enzyme technologies, nucleic acid sequences are synthesized by polymerase chain reaction (PCR) using a nucleic acid template or primer and a polymerase enzyme. One such example is polymerase cycle assembly (PCA). PCA uses PCR to extend short overlapping oligonucleotides into double-stranded sequences. Enzyme synthesis technologies are more suitable for generating longer sequences but are inefficient in generating highly repetitive sequences and sequences with a high GC content.

[0005] Long nucleic acid sequences comprising multiple repeating units have many applications in synthetic biology, diagnostics, and therapy, so it is necessary to identify more efficient methods for their generation.

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 the 3' end of each single-stranded oligonucleotide, the second sequence being located between the first sequence and the third sequence, the first sequences and the third sequences 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 with 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, a step; (ii) enzymatically extending both the first single-stranded oligonucleotide and the second single-stranded oligonucleotide to form a double strand; A method for extending the length of a nucleic acid is provided, which includes the above steps.

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

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

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

[0010] Advantageously, by having a 3’ self-complementary sequence, it is ensured that the sequence is predictably incorporated into the oligonucleotide during elongation, enabling the user to have more control over the order of the sequences within the nucleotide product.

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

[0012] Optionally, the overlapping primer oligonucleotide is a DNA oligonucleotide.

[0013] Optionally, the overlapping primer oligonucleotide is an RNA oligonucleotide.

[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 is different between the first single-stranded oligonucleotide and the second single-stranded oligonucleotide, more diverse sequences can be incorporated into the elongating oligonucleotide by complementary base pairing, and as a result, two separate fourth sequences are incorporated into the nucleic acid sequence.

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

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

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

[0019] Optionally, in addition to being self-complementary, at least one functional site is a palindromic sequence (and can be considered a sequence distinct from the first and third arrays that are self-complementary palindromes).

[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, at least one functional site can be selected from a restriction enzyme site, a capping site, a chemical reactivity modification to a base such as an alkyne, azide, halo modification, a sequence-specific drug binding site, and an enzyme inhibitor site.

[0023] Regarding a functional site that is a chemical reactivity modification to a base, at least one functional site is selected from an alkyne modification, an azide modification, and a halo modification.

[0024] Regarding a functional site that is 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, etc.

[0025] Advantageously, including a restriction site in one of the arrays provides a means to easily cleave the nucleic acid sequence into shorter sequences. For example, sequence A and sequence C may encode two desired oligonucleotides, and sequence B and sequence D may encode two restriction enzyme binding sites. In this example, when a long repetitive sequence is generated according to the method described herein, the associated restriction enzyme can be used to easily shorten the long repetitive sequence into the desired oligonucleotide sequence.

[0026] Optionally, at least one of the first and third sequences includes a tandem repeat.

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

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

[0029] Advantageously, when the first and third sequences include 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 includes at least one modification selected from modified nucleotides, artificial bases, and loop structures.

[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 is particularly desirable in fields such as nanotechnology, biomedicine, and diagnostics.

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

[0033] Optionally, the modified nucleotide may comprise 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP, 5-(octadiynyl)-dCTP, a dye-labeled nucleotide, a quencher-labeled nucleotide, an intrinsically fluorescent nucleotide, an α-phosphate modified nucleotide, γ-[(6-aminohexyl)-imide]-ATPαS, an α,β non-hydrolyzable nucleotide, a β-phosphate modified nucleotide, a β,γ non-hydrolyzable nucleotide, a γ-phosphate modified nucleotide, a non-hydrolyzable dinucleotide, a non-hydrolyzable dye-labeled nucleotide, a biotin-labeled nucleotide, a desthiobiotin-labeled nucleotide, a digoxigenin-labeled nucleotide, a DNP (dinitrophenol)-labeled nucleotide, a photo-labile group-labeled nucleotide, a DBCO-labeled nucleotide, a TCO-labeled nucleotide, a vinyl-labeled nucleotide, a free amino group (-NH2)-labeled nucleotide, a redox dye-labeled nucleotide, a halogen atom-labeled nucleotide, a mercury-labeled nucleotide, a selenium-labeled nucleotide, a ferrocene-labeled nucleotide, a cap analog and derivative, a puromycin analog and derivative, a coenzyme A (CoA) analog and derivative, an NAD analog and derivative, an analog and derivative of a natural RNA nucleobase, a cyclic dinucleotide, a 3’,5’-cyclic nucleotide, a 2’,3’-cyclic nucleotide, a dinucleoside polyphosphate, 6-thiopurine, 7-deazapurine, 7-methylguanosine, a substituted pyrimidine, 5-methylcytidine and related substances, unmodified and modified ddNTP, 2’-fluoro-2’-NTP, 2’-O-methyl-NTP, LNA-NTP, a cleavable base-labeled dNTP, an N1-modified purine, an N6-modified purine, a 6-modified purine, 8-oxoguanosine, 2’-deoxyuridine, 3’-deoxynucleotide, a nucleoside bisphosphate, an alanyl nucleotide, an unmodified purine, a modified dNTP, 3’-O-azidomethyl-dNTP.

[0034] Optionally, the modified nucleotide comprises a linker that attaches the modification to the nucleotide.

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

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

[0037] Preferably, the method (iii) denaturing the double strand to provide the first and second single-stranded polynucleotides, and (iv) annealing the first and second single-stranded polynucleotides to provide an overhang region at at least one of the 5'-ends of the first or second single-stranded polynucleotide, enabling the formation of a polynucleotide double strand between the first single-stranded polynucleotide and the second single-stranded polynucleotide, and (v) enzymatically extending both the first single-stranded polynucleotide and the second single-stranded polynucleotide to form a double strand, and further comprises.

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

[0039] Optionally, the overlapping primer oligonucleotide is immobilized on a surface.

[0040] Preferably, the overlapping primer oligonucleotide is immobilized on the surface by one of the 5'-ends of the first or second single-stranded polynucleotide.

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

[0042] Optionally, the overlapping primer oligonucleotide is immobilized on the surface by a covalent or non-covalent bond.

[0043] Optionally, the overlapping primer oligonucleotide is immobilized on the chemically modified region of the surface.

[0044] Optionally, the overlapping primer oligonucleotide is immobilized on the surface by a linker.

[0045] Optionally, the linker comprises a silane linker molecule, a biotin-streptavidin complex, a thiol-Au linker, an Si-C covalent bond to silicon, an Si-O covalent bond to silicon, an 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 oligonucleotides under appropriate conditions.

[0047] According to a second aspect of the present invention, an overlapping primer oligonucleotide for extending the length of a repetitive sequence of a nucleic acid, the overlapping primer oligonucleotide comprising two partially complementary single-stranded oligonucleotides, each single-stranded oligonucleotide comprising at least first, second and third sequences, the third sequence being located at the 3' end of the first and second single-stranded oligonucleotides, the first sequence and the third sequence being palindromic self-complementary sequences, the second sequence being located between the first sequence and the third sequence, and the third sequence of the first single-stranded oligonucleotide hybridizing with 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 and third sequences 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 can be selected from a restriction enzyme site, a capping site.

[0054] Optionally, at least one of the first and third sequences contains a tandem repeat.

[0055] Optionally, both the first sequence and the third sequence contain 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, a loop structure.

[0058] Optionally, the modified nucleotide is an alkyne-modified nucleotide, an azide-modified nucleotide or a phosphorothioate-modified nucleotide. Optionally, these may include 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP, 5-(octadiynyl)-dCTP.

[0059] Optionally, the modified nucleotide comprises a linker for attaching the modification to the nucleotide.

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

[0061] Optionally, the overlapping primer oligonucleotide is a DNA primer oligonucleotide.

[0062] Optionally, the overlapping primer oligonucleotide is an RNA primer oligonucleotide.

[0063] Further various 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 technical field to which this invention belongs.

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

[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 where a part of one strand is complementary to the other strand and anneals to form a partial duplex, but the full length of the strands is not complementary, and a single-stranded polynucleotide tail occurs 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, and the degree of complementarity is based on the proportion of matching complementary nucleotide pairs. The more nucleotides within a given sequence that are complementary to other sequences, the more stringent the hybridization conditions can be and the more specific the binding of the two sequences becomes. Generally, increasing stringency is achieved by raising the temperature, increasing the ratio of co-solvent, lowering the salt concentration, and other such methods known in the art.

[0068] The term "hybridization conditions" refers to the reagents and reaction conditions (such as temperature, time, etc.) used for hybridization. Generally, the hybridization conditions can be stringent or moderate. The hybridization conditions used in the context of the methods described herein can be either moderate or stringent in order to allow for the formation of mismatched duplexes. Preferably, the hybridization between the unit sequence of the first single-stranded oligonucleotide and the second single-stranded oligonucleotide forms a stable duplex at 65°C or lower. The mismatched duplex is preferably formed at a temperature of up to 65°C, for example 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, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods are described in that reference and either can be used. Preferred examples of stringent hybridization conditions include hybridizing at about 45°C in 6× sodium chloride / sodium citrate (SSC), followed by washing one or more times at 50°C in 0.2× SSC, 0.1% (w / v) SDS. Another example of stringent hybridization conditions includes hybridizing at about 45°C in 6× SSC, followed by washing one or more times at 55°C in 0.2× SSC, 0.1% (w / v) SDS. A further example of stringent hybridization conditions includes hybridizing at about 45°C in 6× SSC, followed by washing one or more times at 60°C in 0.2× SSC, 0.1% (w / v) SDS. Preferably, stringent hybridization conditions are hybridizing at about 45°C in 6× SSC, followed by washing one or more times at 65°C in 0.2× SSC, 0.1% (w / v) SDS. Particularly preferred stringency conditions (and the conditions to be used when the practitioner is unsure what conditions to apply to determine whether a molecule is within the hybridization limitations of the invention) are washing at 65°C in 0.5 molar sodium phosphate, 7% (w / v) SDS, followed by washing one or more times at 65°C in 0.2× SSC, 1% (w / v) SDS.

[0070] The terms "elongation" or "extension" in the context of nucleotide sequences are used interchangeably herein. These refer to the extension of the 3' end and / or 5' end of a polynucleotide by the addition of nucleotides or bases. Chain elongation related to 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 elongating strand hybridizes.

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

[0072] The term "extension conditions" refers to the reagents used and the reaction conditions (e.g., temperature, time, etc.). This describes the extension conditions of the primer polynucleotide. In the present invention, the contact between the mismatched duplex, polymerase, and nucleotide is carried out under extension conditions that allow polynucleotide extension in the 5' to 3' direction. Appropriate extension conditions are known in the art. Preferably, the extension is carried out at a temperature of about 65°C to 75°C for 30 to 120 seconds as needed. Appropriate conditions can be found, for example, in Whitfield C J, Turley A T, Tuite E M, Connolly B A, 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 a nucleotide sequence refers to when one sequence can bind to the other 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 the nucleic acid, and 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 by Saiki et al. (Science 230:1350 - 1354, 1986).

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

[0076] The terms "nucleic acid", "polynucleotide" and "nucleotide" specifically also 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 bases, synthetic bases and modified bases.

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

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

[0079] Embodiments of the present invention will be described by way of example only with reference to the accompanying drawings. In the accompanying drawings, corresponding reference numerals are given to corresponding parts.

Brief Description of the Drawings

[0080]

Figure 1

Figure 2

Figure 3A

Figure 3B

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Figure 5A

Figure 5B

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Figure 7

Figure 8A

Figure 8B

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Figure 10A

Figure 10B

Modes for Carrying Out the Invention

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

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

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

[0084] The overlapping primer oligonucleotide 100 is formed by hybridization of the C sequence of the first SS oligonucleotide 100a and the C sequence of the second SS oligonucleotide 100b. As a result of this hybridization, a partial double strand having an overhang region at the 5' end of each of the SS oligonucleotide sequences 100a, 100b is formed. The overhang region of each SS oligonucleotide comprises both the A and B sequences.

[0085] The repetitive primer oligonucleotide 100 is extended 101 in the presence of a suitable 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'→3' polymerases are available and can be used in the methods described herein. The polymerase preferably has heat stability and high stability such that its activity is substantially retained during the long incubations required for the extension reaction. The polymerase preferably has high processivity. The polymerase preferably does not exhibit non-specific nuclease activity. The polymerase preferably has good fidelity, but can accept various nucleotide analogs as both template and substrate. A high-fidelity polymerase with high-fidelity proofreading activity is not particularly suitable. Preferably, the polymerase lacks 3'→5' exonuclease activity [3'→5'exo(-)], thereby resulting in low fidelity due to the lack of a proofreading function. One of ordinary skill in the art can determine whether a particular polymerase has the required properties as defined above. Exemplary polymerases include, but are not limited to, the 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] The extension of the repetitive primer oligonucleotides can be carried out using a PCR reaction. Thereby, the SS oligonucleotides 100a, 100b in the overhang region are extended to form a stable double strand 107. In this step, the fourth sequence (D) is incorporated into the SS oligonucleotides 100a, 100b. Sequence D is the complementary sequence of sequence B.

[0087] To further extend the sequence, the stable double strand 107 is denatured 102 and re-annealed under appropriate conditions to form a second partial double strand 108. The second partial double strand 108 is formed by the hybridization of the sequence A of the first SS oligonucleotide 100a and the sequence A of the second SS oligonucleotide 100b. As a result of this hybridization, a mismatched double strand having an overhang region at each 5' end of the SS oligonucleotide sequences 100a, 100b is generated. The overhang region of each SS oligonucleotide comprises sequence B, C, and D once and sequence A twice.

[0088] In some embodiments, the sequences are designed to have different melting temperatures (Tms), whereby it is possible to control the amount of mismatches. However, when the Tms are similar, 50% is expected to form mismatched double strands that can be extended.

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

[0090] To further extend the SS oligonucleotides 100a, 100b, the second stable double strand 109 is denatured 104. Further extension can occur under maximum overlap condition 105 or minimum overlap condition 106. Under the maximum overlap condition 105, the SS oligonucleotide 105a is annealed such that there is a maximum amount of overlap between the first and second SS oligonucleotides 100a, 100b. Under the maximum overlap condition 105, the third partial double strand 110 is formed by hybridization of the sequences A, B, C, D, A of the first SS oligonucleotide 100a and the sequences A, D, C, B, A of the second SS oligonucleotide 100b. As a result of this hybridization, a third mismatched double strand 110 with overhang regions at the 5' ends of each of the SS oligonucleotide sequences 100a, 100b is generated. The overhang region of each SS oligonucleotide comprises the sequences A, B, C, D.

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

[0092] The third partial double strand 110 is extended 105b in the presence of a suitable polymerase and nucleotides under extension conditions. Thereby, the SS oligonucleotides 100a, 100b are extended at the overhang regions and the third stable double strand 111 is formed. Further, the sequences A, B, C, and D are incorporated into each of the SS oligonucleotides 100a, 100b, such that as a result, each SS oligonucleotide contains the sequences B, C, and D three times and the sequence A four times.

[0093] Under the minimum overlap condition 106, the SS oligonucleotides are annealed 106a such that the overlap between the first SS oligonucleotide 100a and the second SS oligonucleotide 100b is minimized. Under the minimum overlap condition 106, the fourth partial duplex 112 is formed by hybridization of the sequence A of the first SS oligonucleotide 100a and the sequence A of the second SS oligonucleotide 100b. This hybridization generates a fourth partial duplex 112 having overhang regions at the 5'-ends of each of the SS oligonucleotide sequences 100a, 100b. The overhang region of each SS oligonucleotide comprises the sequences A, B, C, and D twice.

[0094] The fourth partial duplex 112 is extended 106b in the presence of a suitable polymerase and nucleotides under extension conditions. This extends the SS oligonucleotides 100a, 100b of the overhang region and forms the fourth stable duplex 113. Two more of the sequences A, B, C, and D are incorporated into each of the SS oligonucleotides 100a, 100b, such that each SS oligonucleotide contains the sequences B, C, and D four times and the sequence A five times.

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

Example

[0096] (Example 1: 5’ -(AT)5C2(TA)5- 3’ / 3’ -(AT)5C2(TA)5- 5’ ) In one embodiment of the present invention, the starting overlapping primer oligonucleotide 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 each comprised the same sequences A, B, and C. The nucleotide sequences of sequences A, B, and C are shown in Table 1.

[0097]

Table 1

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

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

[0100] In this example, arrays A and C comprise dinucleotide repeat sequences of bases A and T. When arrays 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 arrays B and D in a controlled manner. Advantageously, this enables the user to more finely control the location where the modified bases are incorporated, and thus enables more control over the composition and properties of the extended polynucleotide product. Those skilled in the art will understand that this is merely an example of the present invention, and in other examples, arrays A and C may contain sequences other than dinucleotide repeat sequences. Similarly, those skilled in the art will understand that in other examples, arrays B and D may contain sequences other than mononucleotide repeat sequences.

[0101] Figure 2 shows the ultraviolet absorption of the PCR product after an increase in 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 the DNA concentration increases with an increase in the number of PCR cycles, indicating that new DNA is generated with an increase in the number of PCR cycles.

[0102] Figure 3A shows the visualization of the gel electrophoresis of the PCR product 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 from Figure 3B, after 20 PCR cycles, DNA products with lengths of 1000 - 5000 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 starting repetitive primer oligonucleotide comprised the first and second SS oligonucleotides. The first and second SS oligonucleotides each comprised the same 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 has a sequence in the order of 5’ -ABC- 3’ (SEQ ID 11), and the second SS oligonucleotide has a sequence in the order of 3’ -CBA- 5’ (SEQ ID 12).

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

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

[0108] FIG. 5A shows the visualization of gel electrophoresis 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 FIG. 5B. As can be seen from FIG. 5B, DNA products with a length of 1000 - 5000 base pairs can be detected after 20 PCR cycles.

[0109] (Example 3: 5’ -(AT)5C2(TA)5- 3’ / 3’ -(AT)5C8(TA)5- 5’ ) In another embodiment of the present invention, the starting overlapping primer oligonucleotide comprised a first and a second SS oligonucleotide.

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

[0111]

Table 3

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

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

[0114] During enzyme extension, complementary base pairing occurs and the first and second SS oligonucleotides are extended 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 described in Table 3 above.

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

[0116] Figure 8A shows the visualization of gel electrophoresis 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 from Figure 8B, DNA products with lengths of 1000 - 5000 base pairs can be detected after 20 PCR cycles.

[0117] (Materials and Methods) (Preparation of Duplicate Primer Oligonucleotides) Duplicate 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 HEPES and potassium acetate DNA annealing buffer (480 μL, 1X) in an Eppendorf. The HEPES and potassium acetate buffer is made from 10 mM HEPES, 100 mM KCl, and 1 mM EDTA. The solution was vortexed for several seconds to mix thoroughly, heated at 95 °C for 10 minutes, and then cooled slowly to room temperature (about 25 °C). The duplicate primer oligonucleotides were stored at -20 °C in a freezer when not in use.

[0118] (DNA Extension) 60 μL of Nanopure-H2O was added to 10 μL each of dATP, dTTP, dCTP, and dGTP (all 100 mM) to prepare a 100 μL dNTP mixture at a concentration of 10 mM. For each of the above examples, a primer-specific dNTP mixture was prepared by varying the amount of dNTP added according to the ratio of bases present in the degenerate primer oligonucleotide. For example, the degenerate primer oligonucleotide ((AT)5C2(TA)5 / (AT)5C2(TA)5) used in Example 1 had an A:T:C:G ratio of 5:5:1:1, and the dNTPs were mixed at 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-H2O (60 μL) to prepare 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 by adding nanopure H2O (36 μL) to ThermoPol buffer (5 μL, 10X), degenerate primer oligonucleotide (5 μL, 2 μM), primer-specific dNTP mixture (2.5 μL, 10 mM), and MgSO4 (1 μL, 100 mM). Next, the mixture was vortexed for a few seconds and then added to Deep Vent DNA polymerase enzyme (0.5 μL, 1 U). At 75 °C, 1 unit of Deep Vent DNA polymerase enzyme can incorporate 10 nmol of dNTP. To avoid destroying the polymerase enzyme, the mixture was gently mixed with a pipette tip instead of using a vortex. After 10 cycles, an additional primer-specific dNTP mixture (2.5 μL, 10 mM) was added together with the polymerase enzyme (0.5 μL, 1 U). Then, the Eppendorf was returned to the thermocycler and run for an additional 10 cycles (20 cycles in total) with the same settings. The reagents and products were stored at -20 °C when not in use.

[0120] Each cycle consisted of 1) Denature at 95°C for 30 seconds, and 2) Anneal at 55°C for 30 seconds, and 3) Extend at 72°C for 120 seconds, and It consists of.

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

[0122] In each of the above examples, a different number of cycles was performed. For 20 cycles, the above procedure was followed. For 10 cycles, the first half of the above procedure was performed (i.e., since the experiment was stopped after the first 10 cycles, there was no need for a second addition of dNTP mixture and polymerase enzyme).

[0123] (Purification of Extended DNA Product) For purification, a Monarch® PCR and DNA Cleanup Kit was used. DNA Binding Buffer (100 μL) was added to the extended DNA solution, mixed by pipette, and then the entire sample was transferred to a thick-walled tube and column. The column was centrifuged at 13000 RPM for 1 minute, and then the waste buffer was disposed of as aqueous waste. DNA Wash Buffer (200 μL) was added to the column and this was centrifuged at 13000 RPM for 1 minute. This step was repeated and all the wash solutions were disposed of as aqueous waste. The column was centrifuged again at 13000 RPM for 1 minute to ensure that all the excess liquid was removed and the column was completely dry. That is, all the waste was disposed of as aqueous waste. A 1.5 mL Eppendorf tube was heated to 65°C in a heating block and the column (with DNA attached to the silica) was placed inside. This heating step was for increasing the 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 13000 RPM for 1 minute. The purified extended DNA product was collected in a 1.5 mL Eppendorf tube. All the binding / wash / elution buffers were stored at room temperature.

[0124] (UV / Vis Spectrophotometry) UV / Vis spectra, duplicate primer oligonucleotide concentrations, and purity ratios were recorded using a Thermo Scientific™ NanoDrop™ One Microvolume UV / Vis Spectrophotometer that utilizes surface tension to hold a small amount of sample between two pedestals enabling analysis of 1 μL samples. Most measurements of samples and blanks were recorded using the dsDNA setting. Blanks were obtained using Monarch® DNA Elution Buffer each time before running measurements of each DNA sample. Between each measurement, the pedestals were cleaned using lint-free wipes to minimize cross-sample contamination. The presence of DNA was confirmed by the 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 the data was converted to graphs using Microsoft Excel.

[0126] Example 4: Inclusion of Restriction Sites In another example of the invention, the restriction site is included in the starting duplicate primer oligonucleotide. After extension, the restriction site can be used to cleave the nucleic acid 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 is divided into two parts after digestion with a restriction enzyme, with three bases added to each end of the oligomer.

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

[0130] Treatment with the restriction enzyme yields two double-stranded oligomers of 18 bases and 13 bases, derived from the A / E complementary sequence and the C / D complementary sequence, respectively.

[0131] JPEG2025519607000006.jpg20127

[0132] JPEG2025519607000007.jpg29166

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

[0134] JPEG2025519607000008.jpg22166

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

[0136] The elongation reaction mixture was prepared by adding nanopure H2O (36 μL) to ThermoPol buffer (5 μL, 10X), overlapping primer oligonucleotides (5 μL, 2 μM), primer-specific dNTP mixture (2.5 μL, 10 mM), and MgSO4 (1 μL, 100 mM) in a thin-walled 200 μL Eppendorf. Next, after vortexing the mixture for several seconds, it was added to Deep Vent DNA polymerase enzyme (0.5 μL, 1 U). At 75 °C, 1 unit of Deep Vent (exo-) DNA polymerase enzyme can incorporate 10 nmol of dNTP. To avoid destroying the polymerase enzyme, the mixture was gently mixed with a pipette tip instead of using a vortex. After 10 cycles, primer-specific dNTP mixture (2.5 μL, 10 mM) was added together with polymerase enzyme (0.5 μL, 1 U). Then, the Eppendorf was returned to the thermocycler and run for an additional 10 cycles (total 20 cycles) with the same settings. Reagents and products were stored at -20 °C when not in use.

[0137] Each cycle consisted of 1) denaturing at 95 °C for 30 seconds, 2) annealing at 55 °C for 30 seconds, 3) extending at 72 °C for 120 seconds. After 20 cycles, the mixture was cooled to 4 °C by the thermocycler and held at this temperature for the required period.

[0138]

[0139] For purification, the Monarch® PCR and DNA Cleanup Kit was used. DNA Binding Buffer (100 μL) was added to the extended DNA solution, mixed by pipette, and then the entire sample was transferred to a thick-walled tube and column. The column was centrifuged at 13,000 RPM for 1 minute, and then the waste buffer was disposed of as aqueous waste. DNA Wash Buffer (200 μL) was added to the column and centrifuged at 13,000 RPM for 1 minute. This step was repeated and all wash solutions were disposed of as aqueous waste. The column was centrifuged again at 13,000 RPM for 1 minute to ensure that all excess liquid was removed and the column was completely dry. That is, all waste was disposed of as aqueous waste. A 1.5 mL Eppendorf tube was heated to 60 °C in a heating block and the column (with DNA attached to the silica) was placed inside. This heating step was to increase the 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 / wash / elution buffers were stored at room temperature.

[0140] (UV / Vis spectroscopy) The UV / Vis spectrum, overlapping primer oligonucleotide concentration, and purity ratio, as shown in Figure 9, were recorded using a Thermo Scientific™ NanoDrop™ One microvolume UV / Vis spectrophotometer that uses surface tension to hold a small amount of sample between two pedestals enabling analysis of 1 μL samples. Most measurements of samples and blanks were recorded using the dsDNA setting. Blanks were obtained using Monarch® DNA Elution Buffer each time before performing measurements of each DNA sample. Between each measurement, the pedestals were cleaned using a lint-free wipe to minimize contamination between samples. The presence of DNA was confirmed by the absorption band at 260 nm.

[0141] (Agarose gel electrophoresis) The gel was electrophoresed at 100 V (400 mA) for 60 - 90 minutes. All gels were imaged using UviProMW1 software and a Uvitec Chemiluminescence fluorescence imaging box. All gels were analyzed using ImageJ software and the data was converted to a graph using Microsoft Excel.

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

[0143]

Table 5

[0144] The digested products were run alongside a low range ladder using a 4% MetaPhor agarose gel and the gel electrophoresis was analyzed using Image-J as shown in Figure 10.

[0145] Figure 10(A) shows the visualization of the gel electrophoresis of the 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 extended products of 75 - 300 base pairs.

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

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

[0148] Regarding the use of substantially any plural and / or singular terms in this specification, those skilled in the art can convert from plural to singular and / or from singular to plural according to the context and / or application. In this specification, various singular / plural permutations may be explicitly described for clarity.

[0149] Generally, it will be understood by those skilled in the art that the terms used herein, particularly in the appended claims, are generally intended to be "open" terms (e.g., the terms "comprising" or "including" 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.). Further, those skilled in the art will also understand that if a specific number of introductions to claim descriptions is intended, such intention is explicitly stated in the claims, and if there is no such statement, then such intention does not exist. For example, for the sake of understanding, in the following appended claims, introductory phrases "at least one" and "one or more" may be used to introduce claim descriptions. However, the use of such phrases should not be construed to mean that the introduction of a claim description by the indefinite article "a" or "an" limits a particular claim that includes the introduced claim description to an embodiment that includes only one such description. This is the same even when the introductory phrases "one or more" or "at least one" and the indefinite article "a" or "an" are included in the same claim (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same is true for the use of the definite article used to introduce a claim description. Further, even when a specific number of introduced claim descriptions is explicitly stated, those skilled in the art will recognize that such statement should be interpreted to mean at least the stated number (e.g., a mere statement of "two descriptions" without other modifiers means at least two descriptions, or two or more descriptions).

[0150] Various embodiments of the present disclosure are described herein for purposes of illustration, and it should be understood that various changes can be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, and the true scope is indicated by the appended claims.

Claims

1. A method for extending the length of a nucleic acid, comprising: (i) providing at least one overlapping primer oligonucleotide, wherein the overlapping primer oligonucleotide comprises 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 first, second, and third sequences, the third sequence being located at the 3'-end of each single-stranded oligonucleotide, the second sequence being located between the first sequence and the third sequence, and the first and third sequences 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 with 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; step; (ii) enzymatically extending both the first single-stranded oligonucleotide and the second single-stranded oligonucleotide to form a double strand; A method for extending the length of a nucleic acid, comprising the steps of:

2. 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. The method for extending the length of a nucleic acid according to claim 1.

3. The method for extending the length of a nucleic acid according to claim 1 or 2, wherein the overlapping primer oligonucleotide is a DNA oligonucleotide or an RNA oligonucleotide.

4. The method for extending the length of a nucleic acid according to any one of claims 1 to 3, wherein the second sequence on the first single-stranded oligonucleotide is different from the second sequence on the second single-stranded oligonucleotide.

5. (iii) denaturing the double strand to provide first and second single-stranded polynucleotides; (iv) annealing the first and the second single-stranded polynucleotides to provide an overhang region at at least one of the 5'-ends of the first or the second single-stranded polynucleotide, enabling formation of a polynucleotide double-strand 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 double-strand; The method for extending the length of a nucleic acid according to any one of claims 1 to 4, further comprising the above steps. **Claim 6** The method for extending the length of a nucleic acid according to claim 5, wherein steps (iii) to (v) are repeated to increase the length of the nucleic acid sequence. **Claim 7** The method for extending the length of a nucleic acid according to any one of claims 1 to 6, wherein the overlapping primer oligonucleotide is immobilized on a surface, preferably, the overlapping primer oligonucleotide is immobilized on the surface by one of the 5'-ends of the first or the second single-stranded polynucleotide. **Claim 8** The method for extending the length of a nucleic acid according to claim 7, wherein the surface comprises glass, silica, gold, graphene, graphene oxide, epoxy, plastic, metal, gel matrix, template-stripped metal, or a composite material thereof. **Claim 9** The method for extending the length of a nucleic acid according to claim 7 or 8, wherein the overlapping primer oligonucleotide is immobilized on the surface by a covalent bond or a non-covalent bond, and / or the overlapping primer oligonucleotide is immobilized on a chemically modified region of the surface. **Claim 10** The method for extending the length of a nucleic acid according to any one of claims 7 to 9, wherein the overlapping primer oligonucleotide is immobilized on the surface by a linker, preferably, the linker comprises a silane linker molecule, a biotin-streptavidin complex, a thiol-Au linker, an Si-C covalent bond to silicon, an Si-O covalent bond to silicon, an Si-N covalent bond to silicon, a nanoparticle linker, or a dynamic covalent bond. **Claim 11** The step of providing the at least one overlapping primer oligonucleotide comprises obtaining the first single-stranded oligonucleotide and the second single-stranded oligonucleotide, and hybridizing the first and the second single-stranded oligonucleotides under suitable conditions, a method for extending the length of a nucleic acid according to any one of claims 1 to 10.

12. An overlapping primer oligonucleotide for extending the length of a repetitive sequence of a nucleic acid, wherein the overlapping primer oligonucleotide comprises two partially complementary single-stranded oligonucleotides, and each single-stranded oligonucleotide comprises at least a first, a second and a third sequence, and the third sequence is located at the 3'-end of the first and the 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 with 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 the second single-stranded oligonucleotides, an overlapping primer oligonucleotide for extending the length of a repetitive sequence of a nucleic acid.

13. 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, the overlapping primer oligonucleotide according to claim 12.

14. The first and the third sequences are each at least 6 bases in length, preferably at least 8 bases in length, a method for extending the length of a nucleic acid according to any one of claims 1 to 11 or an overlapping primer oligonucleotide according to claim 12 or 13.

15. The second sequence is at least 2 bases in length, a method for extending the length of a nucleic acid according to any one of claims 1 to 11, 14 or an overlapping primer oligonucleotide according to any one of claims 12 to 14.

16. At least one of the first and third sequences comprises at least one functional site, and the at least one functional site can be selected from a restriction enzyme site, a capping site, a sequence-specific drug binding site, and an enzyme inhibition site. A method for extending the length of a nucleic acid according to any one of claims 1 to 11, 14, 15, or a duplicate primer oligonucleotide according to any one of claims 12 to 15.

17. At least one of the first and third sequences contains a tandem repeat. A method for extending the length of a nucleic acid according to any one of claims 1 to 11, 14 to 16, or a duplicate primer oligonucleotide according to any one of claims 12 to 17.

18. Both the first sequence and the third sequence contain tandem repeats. A method for extending the length of a nucleic acid according to claim 17, or a duplicate primer oligonucleotide.

19. The tandem repeat is a dinucleotide tandem repeat. A method for extending the length of a nucleic acid according to claim 17 or 18, or a duplicate primer oligonucleotide.

20. The second sequence contains at least one modification selected from a modified nucleotide, an artificial base, and a loop structure. A method for extending the length of a nucleic acid according to any one of claims 1 to 11, 14 to 19, or a duplicate primer oligonucleotide according to any one of claims 12 to 19.

21. The modified nucleotide is an alkyne-modified nucleotide, an azide-modified nucleotide, or a phosphorothioate-modified nucleotide, and optionally, these may include 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP, 5-(octadiynyl)-dCTP. A method for extending the length of a nucleic acid according to claim 20, or a duplicate primer oligonucleotide.

22. The modified nucleotide contains a linker for adding the modification to the nucleotide. A method for extending the length of a nucleic acid according to claim 20 or 21, or a duplicate primer oligonucleotide.

23. The loop structure is a G-quadruplex region, a C motif, or an inserted motif DNA. A method for extending the length of a nucleic acid according to claim 20, or a duplicate primer oligonucleotide.

24. The overlapping primer oligonucleotide according to any one of claims 12 to 23, wherein the overlapping primer oligonucleotide is a DNA primer oligonucleotide or an RNA primer oligonucleotide.

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