WO2016170147A1

WO2016170147A1 - Efficiency improving ligation methods

Info

Publication number: WO2016170147A1
Application number: PCT/EP2016/059068
Authority: WO
Inventors: Nan Fang; Katja HEITZ
Original assignee: Qiagen Gmbh
Priority date: 2015-04-22
Filing date: 2016-04-22
Publication date: 2016-10-27

Abstract

The present invention provides new methods and kits to improve the efficiency of ligation reactions, in particular in molecular biology applications, such as the next generation sequencing (NGS) library construction methods and gene cloning. In next-generation sequencing methods, the ligation step is critical in adding sequencing platform-specific adapters to the DNA fragments that are to be sequenced. Said improvement is achieved by the addition of agents, which modulate the melting temperature of dsDNA.

Description

EFFICIENCY IMPROVING LIGATION METHODS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Double-stranded nucleic acids containing blunt ends or cohesive (sticky) ends with an overhang of one or more nucleotides can be joined by means of intermolecular or intramolecular ligation reactions. Examples for the methods for ligating at a specific site are DNA ligation reactions of cohesive ends of DNA fragments, which have been cleaved by a restriction enzyme, or of blunt ends of DNA fragments. Such ligation reactions are commonly used in molecular biology applications, such as next-generation sequencing and gene cloning.

Next-generation sequencing (NGS), also known as high-throughput sequencing allows to acquire genome-wide data using highly parallel sequencing approaches for molecular biology applications, in vitro clinical diagnostics, or for forensics. Such applications include e.g. de novo genome sequencing, transcriptome sequencing and epigenomics, as well as genetic screening for the identification of rare genetic variants and for efficient detection of either inherited or somatic mutations in cancer genes.

Hence, several sequencing platforms have been developed, which allow for low-cost, high-throughput sequencing. Such platforms include lllumina® (Solexa), and Ion torrent: Proton / PGM by Life Technologies/ Thermo Fisher Scientific. NGS technologies, NGS platforms and common applications/fields for NGS technologies are e.g. reviewed in Voelkerding et al. (Clinical Chemistry 55:4 641-658, 2009) and Metzker (Nature

Reviews/Genetics Volume 1 1 , January 2010, pages 31-46).

Three main steps exist in NGS on most current platforms: preparation of the sample for high-throughput sequencing, immobilization on a suitable surface, and the actual sequencing. The preparation step involves random fragmentation of the genomic DNA and addition of adapter sequences to the fragment ends. The commonly used method to generate platform-specific NGS libraries uses multi-step enzymatic reaction protocols to ligate adapters to the DNA fragments to be analyzed.

First, DNA fragments are generated with mechanical, chemical, or enzymatic

fragmentation or by target-specific PCR. Subsequently, the DNA fragments are end- repaired. The end-repair step requires at least two enzymes: (a) a polynucleotide kinase, normally the T4 Polynucleotide Kinase (PNK) that phosphorylates the 5'-terminus of the double stranded DNA fragments; and (b) an enzyme or enzymes with polymerase and exonuclease activities that make the ends of the DNA fragments blunt by either fill-in or trimming reactions, such as e.g. T4 DNA Polymerase. After the end-repair step, for sequencing on platforms, such as those provided by lllumina®, a so-called A-addition step is required, which generates a terminal adenine as a docking site for the

sequencing adapters that have an overhang formed by thymidine nucleotides, i.e. a T- overhang. In this step, an A-overhang is added to the 3'-terminus of the end-repaired PCR product, e.g. by Klenow Fragment exo-, the large fragment of the DNA polymerase I having 5'- 3' polymerase activity, but lacking both 3'- 5' exonuclease activity and 5'- 3' exonuclease activity. Alternatively, the A-addition step can also be facilitated with enzymes having terminal nucleotide transferase activity, such as the Taq polymerase. Following the A-addition step, the sequencing adapter can be ligated to the DNA by a ligase, such as the T4 DNA Ligase. For other sequencing platforms, such as Ion Torrent PGM/ Proton by Life Technologies®, the A-addition step is not required and blunt-ended adapters are ligated by a T4 DNA ligase directly to the end-repaired DNA fragments. The low efficiency of library generation methods can be a draw-back if a sequencing library needs to be constructed from a small amount of input DNA, such as an input amount lower than 1 ng. Most current library construction methods cannot deliver high adaptor-ligation efficiency with such low input amount; therefore, only a small percentage of the input DNA is converted into sequencing library. This could lead to either lack of sufficient library for sequencing, or the library complexity is low and cannot faithfully represent the sequence in the original sample.

Thus, there is a need in the art for sample preparation methods for a NGS library protocol generation and ligation preparation for gene cloning, especially when small amounts of DNA are to be analyzed.

SUMMARY OF THE INVENTION

The present invention relates to agents, which modulate the melting temperature (Tm) of dsDNA in the dsDNA ligation reactions to improve the efficiency and specificity of ligation reactions in molecular biology applications, such as gene library generation and gene cloning. Such ligation reactions may be intermolecular or intramolecular.

In particular, such an improvement is disclosed herein for gene library generation, where the presence of an agent that modulates the melting temperature of dsDNA leads to an enhanced library and specificity. In the context of sequencing library construction, high ligation specificity means that end-repaired DNA fragments and adapters, not DNA fragments or adapters by themselves, are ligated together. This is necessary to prevent sequencing artefacts that can arise from DNA or adapter dimers and concatemers. Surprisingly, the addition of Tm-modulating compounds significantly enhance both the overall yield and the specificity of the ligation product, and hence the overall yield and specificity of a generated NGS library. Similarly, in the presence of said agents in gene cloning, the insert gene DNA and the vector DNA are ligated together, not the insert gene DNA and the vector DNA by themselves. The overall yield increase is at least 70% in the presence of a Tm-modulating agent.

One aspect of the present invention refers to methods of generating dsDNA, wherein the method comprises ligating a first and a second dsDNA, both optionally having one or two single-stranded end(s), in the presence of a DNA ligase and an agent, which modulates the melting temperature of dsDNA.

In some embodiments, the present invention refers to a method for ligating a first and a second ds DNA, wherein both the first and the second dsDNAs comprise two ssDNA regions, whereby each of the ssDNA region ends of the first dsDNA ligates with each of the complementary ss region ends of the second dsDNA to provide ligated circular dsDNA in the presence of a an agent, which modifies the melting temperature of dsDNA. Herein it is preferred that the first or the second DNA is capable of conferring the ability to auto-replicate within competent cells. In other embodiments, the invention relates to a method for generation of a sequencing library, wherein the method comprises the steps of:

(i) providing DNA fragments;

(ii) end-repairing the DNA fragments by a polynucleotide kinase enzyme and an enzyme with polymerase and exonuclease activities to obtain blunt-ended, 5' phosphorylated DNA fragments;

(iii) optionally adding a terminal adenine to the end of the end-repaired DNA fragments by a deoxynucleotidyl transferase enzyme; and

(iv) ligating the DNA fragments, optionally having the terminal adenine, with sequencing adapters wherein preferably the adapters have a terminal thymidine if the fragments have a terminal adenine. In some embodiments, said method further comprises step (v), wherein the ligated fragments of step (iv) are purified and size-selected for sequencing.

In some embodiments, said method further comprises step (vi), wherein the adapter- ligated fragments are amplified and the amplification product is optionally purified prior to sequencing.

In another embodiment, the fragments of step (v) or (vi) are subjected to sequencing. A further aspect of the invention refers to a kit comprising:

(i) a DNA ligase; and

(ii) an agent, which modulates the melting temperature of dsDNA.

In another embodiment, the agent which modulates the melting temperature of dsDNA is selected from any one of tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and Triton®X-100, and mixtures thereof.

In a preferred embodiment, the agent which modulates the melting temperature of dsDNA is selected from any one of tetramethylammonium chloride (TMAC),

piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3, 4,5,6- tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), and mixtures thereof.

In some embodiments, the agent which modulates the melting temperature of dsDNA is in a ligation buffer.

In some embodiments, the ligase and the agent which modulates the melting temperature of dsDNA are in separate containers. In some embodiments, the above kit further comprises:

(i) a polynucleotide kinase and a DNA polymerase; and

(ii) optionally a deoxynucleotidyl transferase.

In the methods or kits referenced above, the polynucleotide kinase enzyme is the T4 Polynucleotide Kinase (PNK) and the enzyme with polymerase and exonuclease activity is the T4 DNA Polymerase; and/or wherein the deoxynucleotidyl transferase enzyme is a Taq polymerase or a Klenow Fragment exo-.

In the methods or kits referenced above, the agent, which modulates the melting temperature of dsDNA, is an organic compound or a biochemical substance.

In the methods or kits referenced above, the organic compound is selected from tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), and Trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4-carboxy- 3,4,5,6-tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and

Triton®X-100, and mixtures thereof.

In another embodiment, in the methods or kits referenced above, the organic compound is selected from tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), and

Trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3, 4,5,6- tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), and mixtures thereof.

Preferably, the agent, which modulates the melting temperature of dsDNA, increases the melting temperature of dsDNA.

More preferably, the compound increasing the melting temperature of dsDNA is tetramethylammonium chloride (TMAC) or tetramethylpiperazinium chloride. In some embodiments, the DNA ligase in any of the above ligations is a T3 DNA ligase or a T4 DNA ligase. In other embodiments, the ligase is a T7 DNA ligase or an

Ampligase®.

In some embodiments of any one of the above methods, each of the first and the second dsDNA have one or two single- stranded DNA (ssDNA) region end(s), wherein this/these ssDNA region end(s) is/are less than 20 nucleotides (nt) in length.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : TMAC improves ligation efficiency. The Agilent Bioanalyzer graphs below show the size distribution and quantity of the sequencing libraries generated with either standard ligation condition (Fig. 1A, "Control") or additional TMAC in the Ligation reaction (Fig. 1 B, "TMAC in Ligation"). The addition of TMAC in the ligation reaction can significantly improve the yield of the library, as determined by the peak heights at about 500 bp, which is the expected size of adapter-ligated sequencing libraries. Figure 2: TMAC improves ligation efficiency. The diagram below shows the

concentrations of the sequencing library, as determined by qPCR. "Control" refers to a standard ligation condition. "TMAC in Ligation", additional TMAC in the ligation reaction.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA may be used. These techniques are well known and are explained in, for example, Current Protocols in Molecular Biology, Volumes 1, 11, and III, 1997 (F. M. Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning; the series, Methods In Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively). The term "high stringency" refers to conditions, under which ability of nucleic acids with certain mismatched bases to hybridize is reduced or completely eliminated. Higher stringency conditions result in a higher ratio of the amount of hybridization of sequences with no mismatches when compared to the amount of hybridization of sequences with one or more mismatches. Preferably, under conditions of "high stringency" only exact matches of bases of nucleotides will anneal and stay together.

The term "agent, which modulates the melting temperature of dsDNA" or "Tm-modulating agent" refers to any organic molecule or biochemical substance, such as an amino acid, oligopeptide (2 to 10 amino acids), polypeptide (at least 10 amino acids to about 100 amino acids), protein, lipid, carbohydrate, or a mixture thereof, which increases or which decreases the melting temperature of dsDNA. Preferably, such a compound increases the melting temperature of dsDNA.

The term "melting temperature of double-stranded nucleic acids" is the temperature, at which half of the DNA strands are in the random coil or single-stranded (ssDNA) state, and half of the DNA strands are in a double-stranded state. Tm depends on the length of the DNA molecule and its specific nucleotide sequence, in particular, the guanine (G) and cytosine (C) content. In this context, the double-stranded nucleic acids refer to dsDNA, dsRNA or RNA:DNA hybrids. The melting temperature also depends on the ionic strength of the solution. One may calculate the melting temperature Tm of any given DNA hybrid as shown:

Tm = 81.5°C + 0.41 (%G + %C) - 550/n n = probe length (number of nucleotides). The equation melting temperature used above refers to the melting temperature that was measured under standard conditions (about 0.8M NaCI, neutral pH (about pH 7.0)).

The term "modulate", and "modify" are used herein as synonyms and are used in the present invention to refer to an increase of the melting temperature of an adenine- thymidine-bond (A:T-bond) or to a decrease of the melting temperature of a guanine- cytosine bond (G:C-bond), which results in an overall increase of the melting

temperature of dsDNA or an overall decrease of the melting temperature of dsDNA, respectively. Experimentally, a melting temperature can be measured assessing dissociation- characteristics of double-stranded DNA during heating.

The terms "next generation sequencing" and "high-throughput sequencing" are used as synonyms.

The term "library" refers to a large number of nucleic acid fragments, here the collection of DNA fragments for sequencing analysis. The libraries referred to herein are generated by fragmentation of a sample to be analyzed, end-repairing, optionally addition of a terminal adenine, and ligation of fragments into adapters. Optionally, the purified DNA fragments are amplified or enriched before they are sequenced. The term "high ligation specificity" means that only the desired DNA, such as insert and vector and not the insert or the vector by themselves, or end-repaired DNA fragments and adapters, not DNA fragments or adapters by themselves, are ligated together. The specificity itself can be measured by methods known to the skilled person, such as PCR.

As used herein, the term "about" when used together with a numerical value (e.g., a pH value or a percentage value) is intended to encompass a deviation of 20%, preferably 10%, more preferably 5%, even more preferably of 2%, and most preferably of 1 % from that value. When used together with a numerical value it is at the same time to be understood as individually disclosing that exact numerical value as a preferred embodiment in accordance with the present invention.

The term "restriction endonuclease" is used herein in its commonly accepted sense as a site specific endodeoxyribonuclease and isoschizomers thereof. Restriction

endonucleases are well-known compounds as is the method of their preparation; see for example Roberts, Critical Reviews in Biochemistry, November 1976, pages 123-164. Representative restriction endonucleases which may be employed in the method of the invention include, but are not restricted to: Alu I, Ava I, Ava II, Bal I, Bam HI, Bel I, Bgl I, Bst E II, Eco R I, Hae II, Hae III, Hinc II, Hind II, Hind III, Hinf I, Hha I, Hpa I, Hpa II, Hph I, Hin 389I, Kpn II, Pst I, Rru I, Sau 3A, Sal I, Sma I, Sst I, Sst II, Tac I, Taq I, Xba I, Xho I and the like, many of which are commercially available (e.g. NEB, Promega, Life Technologies, and Thermo Scientific). Other restriction endonucleases which may be employed and their preparation are listed in e.g. Roberts, pages 127-130.

The term "median fragment size" means that half of the fragments have a longer length and half of the fragments have a shorter length. As used herein, the term "comprising" is to be construed as encompassing both

"including" and "consisting of, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. "nt" is an abbreviation of "nucleotides".

"bp" is an abbreviation of "base pair".

"T4 Polynucleotide Kinase" refers to an enzyme that catalyzes the transfer and exchange of P, from the γ position of ATP to the 5^'-hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3^'-monophosphates.

"T4 DNA Polymerase" refers to an enzyme that catalyzes the synthesis of DNA in the 5^'→3^' direction and requires the presence of template and primer. This enzyme has a 3^'→5^' exonuclease activity which is much more active than that found in DNA

Polymerase I (E. coli). T4 DNA Polymerase does not exhibit 5^'→3^' exonuclease activity.

"Klenow fragment exo-" or "Klenow fragment (3^'→5^' exo-)" refers to an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5^'→3^' exonuclease activity and the 3^'→5^' exonuclease activity.

"Taq polymerase" refers to a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus. The enzyme catalyzes 5'→3' synthesis of DNA, has no detectable 3'→5' exonuclease (proofreading) activity and possesses low 5'→3' exonuclease activity. In addition, Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity, which is often applied in the addition of additional adenines at the 3'- end of PCR products to generate 3^' adenine overhangs. The terms "deoxynucleotidyl transfer", "terminal nucleotide addition" and "terminal nucleotide transfer" are used herein as synonyms. "T3 DNA ligase" refers to an ATP-dependent dsDNA ligase from bacteriophage T3. It catalyzes the formation of a phosphodiester bond between adjacent 5^' phosphate and 3^' hydroxyl groups of duplex DNA. The enzyme joins both cohesive (sticky) and blunt ends. "T4 DNA Ligase" refers to an enzyme that catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA or RNA. This enzyme joins both blunt end and cohesive (sticky) ends.

"T7 DNA Ligase" is an ATP-dependent ligase from bacteriophage T7. This enzyme joins cohesive (sticky) ends and it is suitable for nick sealing. Blunt-end ligation does not occur in the presence of T7 ligase.

"Ampligase®" refers to a DNA Ligase that catalyzes NAD-dependent ligation of adjacent 3^'-hydroxylated and 5^'-phosphorylated termini in duplex DNA structures that are stable at high temperatures. The enzyme that catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA or RNA. This enzyme joins cohesive (sticky) ends. The half-life of Ampligase® is 48 hours at 65°C and more than 1 hour at 95°C. In most cases, the upper limit on reaction temperatures with Ampligase® is determined by the Tm of the DNA substrate. Under conditions of maximal hybridization stringency, nonspecific ligation is nearly eliminated.

The term "ligation buffer" refers to a conventional buffer for DNA ligation known to the skilled person. The ligation buffer can comprise, for example, 50 mM Tris-HCI, 10 mM MgCI₂, 1 mM ATP, 10 mM DTT, and a pH of 7.5 at 25 °C.

The term "PCR" refers to polymerase chain reaction, which is a standard method in molecular biology for DNA amplification.

The term "qPCR" refers to quantitative real-time PCR, a method used to amplify and simultaneously detect the amount of amplified target DNA molecule fragments. The process involves PCR to amplify one or more specific sequences in a DNA sample. At the same time, a detectable probe, typically a fluorescent probe, is included in the reaction mixture to provide real-time quantification. Two commonly used fluorescent probes for quantification of real-time PCR products are: (1 ) non-sequence-specific fluorescent dyes (e.g., SYBR® Green) that intercalate into double-stranded DNA molecules in a sequence non-specific manner, and (2) sequence-specific DNA probes (e.g., oligonucleotides labeled with fluorescent reporters) that permit detection only after hybridization with the DNA targets or after incorporation into PCR products.

The term "DNA" in the present invention relates to any one of viral DNA, prokaryotic DNA, archaeal DNA, and eukaryotic DNA. The DNA may also be obtained from any one of viral RNA, and mRNA from prokaryotes, archaea, and eukaryotes by generating complementary DNA (cDNA) by using a reverse transcriptase.

Agents that modify the melting temperature of dsDNA The melting temperature can be measured experimentally by assessing dissociation- characteristics of double-stranded DNA during heating.

The energy required to break the base-base hydrogen bonding between two strands of DNA is dependent on their length, GC content and their complementarity. With increased heating of a reaction-mixture that contains double-stranded DNA sequences, the amount of double-stranded DNA decreases. The difference between the denaturing conditions of a double-stranded DNA with a completely complementary sequence and the conditions of a double-stranded DNA with an almost complementary but not completely

complementary sequence can be detected as the difference in absorbance change when such double-stranded DNA is denatured into a single-stranded DNA (melting curve) (for example, see I. V. Razlutuskii, L. S. Shlyakhtenko and Yu. L. lyubchenko: Nucleic Acids Research, Vol. 15, No. 16, pp. 6665-6676 (1987)).

Agents that modify the melting temperature can be identified by measuring the melting curve of a DNA sample under (i) standard conditions and under (ii) identical conditions as in (i) including a Tm-modulating agent, and by comparing the measured melting curve data as described above for double-stranded DNA with a completely complementary sequence and the conditions of a double-stranded DNA with an almost complementary but not completely complementary sequence.

The dissociation can be visualized by UV spectroscopy, or by fluorescence

measurements, where a fluorescent dye is used for readout, such as SYBR® Green I, YO-PRO-I®, or ethidium bromide.

For example, SYBR green fluoresces with 1000-fold higher intensity, when it intercalates into the minor groove of dsDNA.

Alternatively, dual hybridization probes may be used, whereby said probes attach to single-stranded DNA. Two probes are designed to hybridize to adjacent sequences of the target DNA. The probes are labeled with a pair of dyes that allow for fluorescence resonance energy transfer (FRET). The donor dye is e.g. attached to the 3' end of the first probe, while the acceptor dye is attached to the 5' end of the second probe.

The agent, which modulates the melting temperature in dsDNA is an organic compound or a biochemical substance. Compounds that have been identified to modulate the melting temperature of dsDNA and thereby improving efficacy and specificity of DNA ligation include, but are not restricted to compounds such as tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), and trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3, 4,5,6- tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and Triton®X-100, and mixtures thereof.

Preferably, the organic compound increases the melting temperature of dsDNA. Such compounds include, but are not restricted to quaternary ammonium salts having the structure NR₄ ⁺. R may be an alkyl, cycloalkyl or an aryl group. Preferably, such quaternary ammonium salts are tetramethylammonium chloride (TMAC) or

tetramethylpiperazinium chloride. Methods

The present invention refers to ligation methods, in particular to gene cloning methods and methods of generating sequencing libraries.

In particular, the method referred herein is characterized in that the ligation step efficiency and specificity is increased by applying an agent to a ligation reaction, which modulates the melting temperature of dsDNA. Such a ligation step is a critical step in both gene cloning and in generating next generation sequencing libraries. In preferred embodiments, the ligation is carried out in the presence of an agent or more than one agent, which increase(s) the melting temperature of dsDNA.

Next generation sequencing

In some embodiments, the invention relates to a method of generating a sequencing library, wherein the method comprises the steps of:

(i) providing DNA fragments;

(ii) end-repairing the DNA fragments by a polynucleotide kinase enzyme and an enzyme with polymerase and exonuclease activities, preferably DNA polymerase, to obtain blunt- ended, 5' phosphorylated DNA fragments;

(iv) ligating the DNA fragments, optionally having the terminal adenine base, with sequencing adapters wherein preferably the adapters have a terminal thymidine if the fragments have a terminal adenine.

In preferred embodiments, the ligation is carried out under high stringency conditions. In some embodiments, said method further comprises step (v), wherein the ligated fragments of step (iv) are purified and size-selected for sequencing. In some embodiments, said method further comprises step (vi), wherein the adapter- ligated fragments are amplified and the amplification product is optionally purified prior to sequencing. The library fragments of step (v) or step (vi) are subsequently sequenced by using sequencing platforms known to the person skilled in the art, such as lllumina® (Solexa) and Ion Torrent: Proton / PGM by Life Technologies/Thermo Fisher Scientific or other suitable high-throughput sequencing platforms.

The size of the DNA fragment length is a key factor for gene library construction and for sequencing. Typical median lengths of DNA fragments for NGS libraries are between about 150 bps and about 1000 bps, preferably between about 150 bps and about 600 bps, more preferably between about 200 bps and about 500 bps. Most preferably, the median length is about 200 bps, about 300 bps, or about 500 bps.

The preferred amount of DNA starting material for generating a NGS sequencing library and for subsequent sequence analysis ranges from about 1 pg to about 1 μg, preferably from about 10 pg to about 1 μg, and more preferably about 10 pg to about 1 ng. For genomic DNA analysis, the amount of starting material is preferably about 1 pg to about 1 μg, preferably from about 10 pg to about 1 μg, and more preferably about 10 pg to about 1 ng.

In some embodiments, the fragmentation step is mechanical. Preferably, the mechanical fragmentation is among others achieved by ultrasonic acoustic shearing, nebulization forces, sonication, hydrodynamic shearing (e.g. in French pressure cells or by needle shearing). More preferably, specific median fragment length sizes of DNA can be prepared e.g. by ultrasonic acoustic shearing, such as Adaptive Focused Acoustics (AFA)™ by using a Covaris® instrument, according to the manufacturer's instructions. In some embodiments, the fragmentation of DNA step is chemical. Chemical shear may also be employed for the breakup of long RNA fragments. This is typically performed through heat digestion of RNA with a divalent metal cation (magnesium or zinc). The length of the RNA (1 15 bp-350 bp) can be adjusted by increasing or decreasing the time of incubation. In some embodiments, the fragmentation step is enzymatic. Preferably, said enzymatic fragmentation is achieved by digestion of DNA by an endonuclease. Such endonucleases are described in more detail in the Definitions section. Preferably, the fragmentation may also be carried out by employing a transposase known to the person skilled in the art. When applying enzymes for the fragmentation reaction, said fragmentation step may be inactivated by heat.

Step (ii), the end-repair step, is carried out by an enzyme or two enzymes with (a) polynucleotide kinase activity (PNK) and (b) an enzyme with polymerase and exonuclease activities, whereby the exonuclease activity makes the ends of the DNA blunt by fill-in or trimming reactions. Preferably, the enzymes of step (ii) comprise a T4 Polynucleotide Kinase (PNK) and a T4 DNA Polymerase.

Step (iii), the A-addition step, is carried out by an enzyme, which generates an adenine docking site for adapters that have a thymidine overhang (T-overhang). Preferably, the enzyme of step (iii) is a Taq polymerase or Klenow Fragment exo-, the large fragment of the DNA polymerase I having 5'- 3' polymerase activity but lacking both 3'- 5' exonuclease activity and 5'- 3' exonuclease activity. In some preferred embodiments, the enzyme of step (iii) is a thermostable polymerase, preferably a Taq polymerase.

Step (iv), the ligation step for gene library generation, joins either blunt or cohesive (sticky) ends of DNA fragments with either blunt or cohesive (sticky) ends of adapter molecules. Successful ligation of cohesive (sticky) ends requires complementary sequences.

In some embodiments, the length of the nucleotide sequences of the ssDNA region ends of dsDNA for the ligation methods in gene library generation referred to above is less than 20 nt or less than 12 nt, preferably the sequence length is less than 10 nt or less than 8 nt, more preferably 1-6 nt or 1-5 nt. In preferred embodiments, the ssDNA length is 1 nt. In embodiments, where the ssDNA region is 1 nt, ssDNA region of one DNA comprises a terminal (3^') adenine (A) and a the complementary ssDNA of the other DNA comprises a terminal (3^') thymidine (T). Alternatively the terminal ssDNA regions are (3^') cytosine (C) and the complementary terminal (3^') guanine (G).

In preferred embodiments, a fragment comprising terminal, i.e. 3' adenine overhangs serves as a docking site for the sequencing adapters, which comprise a complementary terminal, i.e. 3' thymidine overhang. By using such TA cloning it is not necessary to design a specific pair of primers for each DNA fragment to be analyzed. The same primers can be used for amplification of different templates provided that each template is modified by addition of the same universal primer-binding sequences to its 5' and 3' ends. The adapter sequence can therefore be any DNA fragment of interest, as long as it has a 3' thymidine overhang. The efficiency of the ligation step regarding specificity and library yield is improved by the addition of a molecule, which modulates the melting temperature of dsDNA (Tm modulator). Such Tm-modulators can include any organic molecule or biochemical substance that induces a change to the melting temperature of the DNA. In preferred embodiments, the Tm of the A-T bond is increased, so that the ligation through the A/T overhang is used e.g. by the library construction protocols for lllumina sequencing instruments.

In the methods referenced above, the organic compound is selected from any one of the following: quaternary ammonium salts, such as tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3, 4,5,6- tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and Triton®X-100, and mixtures thereof. Preferably, in the methods referenced above, the organic compound is selected from any one of the following: quaternary ammonium salts, such as tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4- carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3, 4,5,6- tetrahydropyrimidine THP(B), and mixtures thereof. Preferably, the organic compound increases the melting temperature of dsDNA. Such compounds include, but are not restricted to quaternary ammonium salts having the structure NR₄ ⁺. R may be an alkyl, cycloalkyl or an aryl group. Preferably, such quaternary ammonium salts are tetramethylammonium chloride (TMAC) or tetramethylpiperazinium chloride.

The concentration of the above compounds in the above methods is about:

50 mM-5 M, preferably 100 mM-3.5 M, more preferably 500 mM-3.3 M, even more preferably 1 M-3.3 M TMAC;

50 mM-5 M, preferably 100 mM-3.5 M. more preferably 500 mM-3.3 M, even more preferably 1 M-3.3 M tetraethylammonium chloride (TEAC);

50 mM-5 M, preferably 100 mM-3.5 M. more preferably 500 mM-3.3 M, even more preferably 1 M-3.3 M piperazinium chloride;

50 mM-5 M, preferably 100 mM-3.5 M. more preferably 500 mM-3.3 M, even more preferably 1 M-3.3 M tetramethylpiperazinium chloride;

>50mM trimethylamine N-oxide (TMANO);

0.5-3.5 M 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A));

0.5-4 M 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B));

0.1-1 % non-ionic detergents.

In embodiments, where TMAC or tetramethylpiperazinium chloride is the modulating agent, the concentration thereof is 50 mM-5 M, preferably 100 mM-3.5 M, more preferably 500 mM-3.3 M, and even more preferably 1 M-3.3 M. Test PCR reactions (under identical conditions, but with or without the Tm-modulating agent) may be performed to determine the optimum concentration of each of the above mentioned additives, whereby optimal concentration is the concentration, at which the dsDNA has the highest Tm.

Preferably, the ligation enzyme referred to above, in particular the enzyme of step (iv) is a T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, an Ampligase®, or an E. coli DNA- ligase, whereby the T7 DNA ligase, the Ampligase® and the E. coli DNA-ligase only ligate cohesive (sticky) DNA. In embodiments, where cohesive (sticky) end ligation, such as AT-ligation is envisioned, it is preferable to use T7 DNA ligase or an Ampligase®. For cohesive (sticky) end ligation under high stringency conditions Ampligase® is preferred, as its exceptional thermostability reduces the hybridization of mismatched base pairs. Preferably, step (iv) comprises T4 DNA ligase when blunt ends are to be ligated.

The ligation step is carried out at 4-50°C, depending on the optimal temperature for the ligase^'s activity. For T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, and E.coli DNA ligase, the preferred ligase temperature is 4-25°C. In embodiments, where Ampligase® is used, the ligation temperature is adapted according to the Tm of the DNA substrate to be ligated.

In gene library generation methods, after generating ligated fragments, said fragments are purified and size-selected on e.g. silicon containing surface of a binding matrix in the presence of a salt, preferably a chaotropic salt. The size of DNA molecules that bind to the binding matrix can be controlled e.g. by the salt concentration or the pH value of the binding mixture. Such purification is e.g. described in WO 2014/122288 A1. Suitable columns for such a size selection method include the GeneRead™ Size Selection Kit. A further DNA size selection method includes agarose gel electrophoresis. The purified fragments may be used directly for subsequent sequencing. Alternatively, prior to the sequencing step, the purified fragments may be amplified for library enrichment by PCR- based methods known to the person skilled in the art, or by capture-by-hybridization, i.e. on-array or in-solution hybrid capture; or by capture-by-circularization, i.e. molecular inversion probe-based methods. Preferably, library enrichment is carried out by PCR amplification. In some embodiments, the ligation reaction in gene library generation is characterized in that the first dsDNA used in such ligation reactions comprises ssDNA regions at both of its termini, which may or may not be identical. Preferably, such terminal ssDNA regions are identical. More preferably, each of the terminal ssDNA regions comprises a terminal adenine. Each of the termini hybridizes under high stringency conditions with a complementary ssDNA region of a second dsDNA, respectively. Preferably, such a second dsDNA is a sequence adaptor. More preferably, such a sequence adaptor comprises a terminal thymidine. Gene Cloning

In some embodiments, a ligation method for gene cloning of two dsDNA (intermolecular) is disclosed, each comprising two ssDNA regions, whereby the two ssDNA regions in one dsDNA may be identical or non-identical. The terminal ssDNA regions of the first and the second dsDNA hybridize under high stringency conditions in the presence of Tm-modulating agent. Preferably, the ssDNA ends of the dsDNA to be ligated are complementary. In more preferred embodiments, the ligation is carried out in the presence of Tm-modulating agent, which increases the melting temperature of dsDNA. In gene cloning methods, each of the ssDNA region ends of the first dsDNA ligates with each of the ss region ends of the second dsDNA to provide ligated circular dsDNA in the presence of an agent, which modulates the melting temperature of dsDNA, preferably, such an agent increases the melting temperature of dsDNA. In preferred embodiments, the first DNA or the second DNA is capable of conferring the ability to auto-replicate within competent cells. The use of ligating nucleic acids in the presence or an agent, which modulates the melting temperature of DNA results in an increased number of transformed host cells after transformation with the ligated molecules with chemically transformed host cells or with host cells transformed by electroporation. A ligation yield increase may also be assessed by methods known to the skilled person, such as agarose gel electrophoresis. The nucleotide sequence length of the DNA for ligation reactions, in particular gene cloning, more particular in vitro gene cloning is not restricted, as long as it agrees with the objective of this invention and accomplishes the functional effects of the invention. The appropriate scope of the aforementioned length can be understood by a person skilled in the art in the field of molecular biology.

The ratio of DNAs to be ligated is not restricted, and may be any, as long as they are within a range that does not adversely affect the correct ligation of each end. In embodiments for cloning of a specific gene, it is preferable to use the DNA to be ligated in a concentration that is equimolar to the DNA comprising the whole or partial gene. Other ratios of a vector and a gene to be inserted are 1 :2, 1 :5, 1 : 10, and 1 :20. More preferably, such a ratio is 1 :5.

When ligating two dsDNA, such as vector DNA and insert gene DNA, the vector DNA is preferably a DNA that can be introduced into a suitable competent cell, wherein it can auto-replicate.

Such vectors are selected according to the competent cells into which the ligate is introduced. For example, for E.coli, the commercially available vectors or plasmids can be used. Such vectors include, but are not restricted to pBR322, pQE series (N-terminus vectors: pQE-9, pQE-30, pQE31 , pQE-32, and pQE-40; C-terminus vectors: pQE16, pQE60, pQE-70 (Qiagen), and pUC series (for example, pUC18, pSP64, pGEM-3, pBluescript). When using yeast as said cells, such vectors include, but are not restricted to Yep24, Ylp5. When using Bacillus, such vectors include, but are not restricted to pHY300 and PLK. Insect cell expression vectors include, but are not restricted to Easy Xpress plX3.0 and pIX 4.0 (Qiagen). Vectors for E.coli, insect cell, and mammalian cell expression include, but are not restricted to pQE Trisystem vectors (Qiagen).

The efficiency of the ligation step regarding specificity and library yield is improved by the addition of a molecule, which modulates the melting temperature of dsDNA (Tm modulator). Such Tm-modulators can include any organic molecule or biochemical substance that induces a change to the melting temperature of the DNA. In preferred embodiments, the Tm of the A-T bond is increased, so that the ligation through the A/T overhang is used e.g. by the library construction protocols for lllumina sequencing instruments. The organic compound is selected from any one of the following: quaternary ammonium salts, such as tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and Triton®X-100, and mixtures thereof.

Preferably, in the methods referenced above, the organic compound is selected from any one of the following: quaternary ammonium salts, such as tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride,

tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4- carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3, 4,5,6- tetrahydropyrimidine THP(B), and mixtures thereof.

tetramethylpiperazinium chloride. The concentration of the above compounds in the above methods is about:

50 mM-5 M, preferably 100 mM-3.5 M. more preferably 500 mM-3.3 M, even more preferably 1 M-3.3 M piperazinium chloride; 50 mM-5 M, preferably 100 mM-3.5 M. more preferably 500 mM-3.3 M, even more preferably 1 M-3.3 M tetramethylpiperazinium chloride;

>50mM trimethylamine N-oxide (TMANO);

0.5-3.5 M 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A));

0.5-4 M 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B));

0.1-1 % non-ionic detergents.

In embodiments, where TMAC or tetramethylpiperazinium chloride is the modulating agent, the tetramethylammonium or tetramethylpiperazinium chloride concentration is 50 mM-5 M, preferably 100 mM-3.5 M, more preferably 500 mM-3.3 M, and even more preferably 1 M-3.3 M.

Test PCR reactions (under identical conditions, but with or without the Tm-modulating agent) may be performed to determine the optimum concentration of each of the above mentioned additives, whereby optimal concentration is the concentration, at which the dsDNA has the highest Tm.

Preferably, the DNA ligation enzyme referred to above is a T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, an Ampligase®, or an E. coli DNA-ligase, whereby the T7 DNA ligase, the Ampligase® and the E. coli DNA-ligase only ligate cohesive (sticky) DNA. In embodiments, where cohesive (sticky) end ligation, such as AT-ligation is envisioned, it is preferable to use T7 DNA ligase or Ampligase®. For cohesive (sticky) end ligation under high stringency conditions Ampligase® is preferred, as its exceptional thermostability permits very high hybridization stringency and ligation specificity. T4 DNA ligase is preferred when blunt ends are to be ligated.

The ligation step is carried out at 4-50°C, depending on the optimal temperature for the ligase^'s activity. For T3 DNA ligase, T4 DNA ligase, T7 DNA ligase and E.coli DNA ligase, the preferred ligase temperature is 4-25°C. In embodiments, where Ampligase® is used, the ligation temperature is adapted according to the Tm of the DNA substrate to be ligated. Kits

Another aspect of the invention relates to kits comprising:

(i) a DNA ligase; and

(ii) an agent, which modulates the melting temperature of dsDNA.

In the kits referenced above, the Tm-modulating agent is an organic compound or a biochemical substance. Preferably, the organic compound increases the melting temperature of dsDNA. Such compounds include, but are not restricted to quaternary ammonium salts having the structure NR₄ ⁺. R may be hydrogen, an alkyl, cycloalkyl or an aryl group. Preferably, such quaternary ammonium salts are tetramethylammonium chloride (TMAC) or tetramethylpiperazinium chloride.

In some embodiments, the agent which modulates the melting temperature of dsDNA is selected from any one of tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and Triton®X-100, and mixtures thereof.

In a preferred embodiment, the agent, which modulates the melting temperature of dsDNA, is selected from any one of tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3, 4,5,6- tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), and mixtures thereof. In some embodiments, said invention relates to kits additionally comprising:

(i) a polynucleotide kinase and an enzyme, with polymerase and exonuclease activities, preferably DNA polymerase; and (ii) optionally a deoxynucleotidyl transferase;

In a preferred embodiment, in the disclosed kits, the ligase and the agent which modulates the melting temperature of dsDNA are in separate containers.

In preferred embodiments of the kits, the polynucleotide kinase enzyme is the T4 Polynucleotide Kinase (PNK) and the enzyme with polymerase and exonuclease activity is the T4 DNA Polymerase and/or the deoxynucleotidyl transferase enzyme is a Taq polymerase or a Klenow Fragment exo-.

The DNA ligase is a T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, an Ampligase®, or an E. coli DNA-ligase, whereby the T7 DNA ligase, Ampligase® and the E. coli DNA- ligase only ligate cohesive (sticky) DNA. Therefore, more preferably, step (iv) comprises T4 DNA ligase when blunt ends are to be ligated. For cohesive (sticky) end ligation in step (iv), T7 DNA ligase or Ampligase® is preferred. For cohesive (sticky) end ligation under high stringency conditions Ampligase® is preferred as its exceptional thermostability permits high hybridization stringency and ligation specificity.

The concentration of the above compounds in the above kits is about:

>50mM trimethlamine N-oxide (TMANO);

0.5-3.5 M 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A));

0.5-4 M 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B));

0.1-1 % non-ionic detergents. In embodiments, where TMAC is the modulating agent, the tetramethylammonium or tetramethylpiperazinium chloride concentration is 50 mM-5 M, preferably 100 mM-3.5 M, more preferably 500 mM-3.3 M, and even more preferably 1 M-3.3 M.

In preferred embodiments, such kits are used for gene cloning.

In other embodiments, such kits are used for generating a sequencing library.

EXAMPLES gDNA from E.coli DH10B is sheared to an average fragment size of 300 bp (Covaris S220 Focused-ultrasonicator, Covaris), and 10 pg of sheared DNA is used for each library construction test. GeneRead™ DNA Library Prep I Core Kit, GeneRead™ DNA I Amp Kit, GeneRead™ Adapter I Set 12-Plex (72), and GeneRead™ Size Selection Kit (all from QIAGEN) are used according to manufacturer's instructions with the following modifications: 0.5 U of the Taq polymerase (QIAGEN) and 0.5 mM of dATP (QIAGEN) are added to the end-repair reaction; the temperature profile for end-repair reaction is 30 minutes at 25°C, and 30 minutes at 72°C, where the 72°C step was used to both inactivate end-repair enzymes and utilize the terminal transferase activity of the Taq enzyme to add an adenine to the 3' of the DNA fragments. The separate A-addition step using Klenow fragment (3'- 5' exo-) is therefore removed from the protocol.

0.05 μΜ of sequencing adapter was used in the ligation steps.

Following the ligation step, the library was first purified with the GeneRead Size

Selection Kit (QIAGEN), then amplified for 22 cycles with adapter-specific primers in PCR (GeneRead DNA I Amp Kit, QIAGEN), and purified again with GeneRead Size Selection Kit (QIAGEN). The final sequencing libraries were qualified with Agilent Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent) and quantified with the qPCR method (QuantiFast Sybr Green Kit, QIAGEN). The sequencing libraries were also sequenced on a miSeq instrument (lllumina) using a dual-layer 300 nt Flow Cell and lllumina® miSeq Reagent Kit V2 (300). Sequencing data were analyzed with CLC Genomics Workbench software (QIAGEN).

As a proof of principle, either standard ligation condition was used or TMAC was added at the final concentration of 0.1 mM in the ligation step. As shown in Figure 1 and Figure 2, both Agilent Bioanalyzer and qPCR results demonstrated that the addition of the TMAC into the ligation reaction ("TMAC in Ligation") could significantly increase the library yield compared to the ligation reaction without additional TMAC ("Control"). Furthermore, sequencing data showed that the addition of the TMAC in the sequencing significantly reduces the number of regions with zero coverage. This demonstrated that the addition of TMAC in the ligation reaction enables a higher percentage of the genomic DNA regions to be ligated to the sequencing adaptors and sequenced.

Example 1 :

The above amplified product of the test and control samples was qualitatively analyzed by using Agilent Bioanalyzer and High Sensitivity DNA Analysis Kit (Agilent).

Example 2:

The above amplified product of the test and control samples was quantitatively analyzed by using qPCR method (QuantiFast Sybr Green Kit, QIAGEN).

Example 3:

The sequencing libraries constructed with or without TMAC were sequenced. Sequence data were mapped to the E.coli DH10B. The number of Zero Coverage Regions, which reflected the drop-out of genomic regions during library construction and in turn sequencing, was significantly reduced by the addition of TMAC in the ligation (from 127 Zero Coverage Regions No TMAC TMAC

Count 127 28

Minimum length 1 1

Maximum length 115 82

Mean length 23,05 24,11

Standard deviation 20,9 21,19

Total length 2.927 675

Table 1 : Zero Coverage Regions in sequencing for the library generated with or without TMAC in ligation reaction.

Claims

A method of generating double-stranded DNA (dsDNA), wherein the method comprises ligating a first and a second dsDNA, both optionally having one or two single-stranded end(s), in the presence of a DNA ligase and an agent, which modulates the melting temperature of dsDNA, wherein the agent is selected from any one of tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3, 4,5,6- tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and Triton®X-100, and mixtures thereof.

The method of claim 1 , wherein the agent, which modulates the melting temperature of dsDNA is selected from any one of tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2- methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4- carboxy-3,4,5,6-tetrahydropyrimidine THP(B), and mixtures thereof.

The method of claims 1 or 2, wherein the method comprises the steps of:

(i) providing DNA fragments;

(ii) end-repairing the DNA fragments using a polynucleotide kinase and an enzyme with polymerase and exonuclease activities to obtain end-repaired DNA fragments;

(iii) optionally adding a terminal adenine to the end of the end-repaired DNA fragments using a deoxynucleotidyl transferase enzyme; and

(iv) ligating the DNA fragments, optionally having the terminal adenine, with sequencing adapters, wherein preferably the adapters have a terminal thymidine if the fragments have a terminal adenine. The method of claim 3, further comprising step (v), wherein the ligated fragments of step (iv) are purified and size-selected.

The method of claim 4, further comprising step (vi), wherein the adapter-ligated fragments are amplified and the amplification product is optionally purified.

A kit comprising:

(i) a DNA ligase; and

(ii) an agent, which modulates the melting temperature of dsDNA, wherein the agent is selected from any one of tetramethylammonium chloride (TMAC), piperazinium chloride, tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5- hydroxy-3,4,5,6-tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3, 4,5,6- tetrahydropyrimidine THP(B), non-ionic detergents, such as NP-40, and

Triton®X-100, and mixtures thereof.

The kit of claim 6, wherein the agent is selected from any one of

tetramethylammonium chloride (TMAC), piperazinium chloride,

tetramethylpiperazinium chloride, tetraethylammonium chloride (TEAC), trimethylamine N-oxide (TMANO), 2-methyl-4-carboxy-5-hydroxy-3, 4,5,6- tetrahydropyrimidine THP(A), 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine THP(B), and mixtures thereof.

The kit of claims 6 or 7, wherein the ligase and the agent are in separate containers.

The kit of claims 6 to 8, wherein the kit further comprises:

(i) a polynucleotide kinase and an enzyme with polymerase and exonuclease activities; and

(ii) optionally a deoxynucleotidyl transferase.

The method of any one of claims 3-5, or the kit of claim 9, wherein (i) the polynucleotide kinase is the T4 Polynucleotide Kinase (PNK);

(ii) the enzyme with polymerase and exonuclease activity is the T4 DNA

Polymerase; and/or

(iii) the deoxynucleotidyl transferase enzyme is a Taq polymerase or a Klenow Fragment exo-.

1 1 . The method of claims 1 or 2, wherein each of dsDNAs comprises two single- stranded DNA ends, whereby each of the ss region ends of the first dsDNA ligates with each of the complementary ssDNA region ends of the second dsDNA, respectively, to provide ligated circular dsDNA, wherein preferably the first or the second DNA is capable of conferring the ability to auto-replicate within competent cells.

12. The method of any one of claims 1-5 and 10-1 1 , or the kit of any one of claims 6-9, wherein the agent is tetramethyl ammonium chloride (TMAC) or tetramethylpiperazinium chloride.

13. The method of any one of claims 1-5 and 10-12, or the kit of any one of claims 6-9, wherein the DNA ligase is a T3 DNA ligase or a T4 DNA ligase.

14. The method of any one of claims 1-5 and 10-12, or the kit of any one of claims 6-9, wherein the DNA ligase is a T7 DNA ligase or an Ampligase®.

15. The method of any one of claims 1-5 and 10-14, wherein each of the one or two single-stranded DNA (ssDNA) region end(s), is/are less than 20 nucleotides (nt) in length.