Patent Application
20110224106
The present invention is directed to the field of analysis of nucleic acids and more particularly to methods for producing single-stranded circular nucleic acid for improving the efficiency of amplification and sequencing technologies.
Single-stranded circular DNA has been found to be useful in many different areas of biotechnology. One important use is as a substrate for rolling circle DNA replication. In this procedure, a single-stranded circle of DNA is mixed with a short strand of single-stranded complementary primer DNA and the two separate strands are allowed to anneal. After addition of a DNA polymerase, such as the Klenow fragment, the intact circle is used as a template by the enzyme and then replicated from the 3′-end of the primer strand. After the enzyme has gone around the circular template, it encounters the 5′-end of the primer, which is then displaced from the template strand so that the enzyme continues to move around the circular template while a long, unbroken single strand of DNA is generated. Such single strand has been referred to as single-stranded concatenated DNA. Single-stranded circular products are ideally suited for use as a substrate in such processes. Such products can ultimately yield single-strand concatenated DNA having numerous different sequential segments that can act as probes, detection sites or restriction sites for further processing.
Libraries of single-strand circular DNA are also useful for rapid DNA sequencing methods. Single molecule real time (SMRT) DNA sequencing technologies have the capability to re-sequence a single segment of double-stranded DNA repeatedly by using a single-strand circular DNA molecule as a template. Pyrosequencing is another rapid sequencing method which would benefit from the use of libraries of single-strand circular DNA (SMRT and pyrosequencing methods are described, for example in PCT publication WO2009120372 which is incorporated herein by reference in entirety).
The present invention is directed to methods for producing single-stranded circular nucleic acid.
An object of the invention is to provide a rapid and unbiased method for generating single-stranded circular nucleic acids for use as templates in applications such as nucleic acid sequencing and rolling circle amplification of nucleic acids.
A further object of the invention is to provide a method for generating a library of single-stranded circular nucleic acid molecules.
A further object of the invention is to provide a method for generating single-stranded circular nucleic acid molecules using simplified processes which are amenable to adaptation within microfluidics devices.
A further object of the invention is to provide a kit for use in producing single-stranded circular nucleic acid and libraries thereof.
A further object of the invention is to provide a system for producing single-stranded circular nucleic acid and libraries thereof.
The methods disclosed herein employ hairpin polynucleotides and a transposase to fragment nucleic acid segments and ligate hairpin polynucleotides to the fragments. A polymerase is then used to fill in the nucleobase segment gaps formed by the fragmentation process. These methods eliminate the need for fragmenting genomic nucleic acid and waiting for slow ligation reactions. The random nucleic acid integration reactions catalyzed by certain transposases provide unbiased generation of fragments.
A method is provided for generating single stranded circular nucleic acid from a sample of target nucleic acid. A complex comprising a transposase and a plurality of hairpin polynucleotides is formed with each of the hairpin polynucleotides having a duplex region comprising a transposase recognition sequence. The complex is mixed with the target nucleic acid, thereby fragmenting the target nucleic acid and ligating the hairpin polynucleotides to the target nucleic acid to form hairpin-linked nucleic acid fragments, each having a nucleobase segment gap between each fragment and its corresponding hairpin polynucleotide. The hairpin-linked fragments are contacted with a ligase, thereby ligating the hairpin-linked fragments together to form single-stranded circular nucleic acid comprising a pair of opposing loops and an intervening duplex region comprising a pair of nucleobase segment gaps. The single-single stranded circular nucleic acid is then contacted with a polymerase and nucleotide triphosphates, thereby filling the nucleobase segment gaps.
In another aspect, a method is provided for preparing a library of single-stranded circular nucleic acid which represents a genome of a virus or organism. A complex is formed comprising a transposase and a plurality of hairpin polynucleotides, each having a duplex region comprising a transposase recognition sequence. The complex is mixed with nucleic acid representing the genome, thereby fragmenting the nucleic acid and ligating the hairpin polynucleotides to the nucleic acid to form hairpin-linked nucleic acid fragments, each having a nucleobase segment gap between each fragment and its corresponding hairpin polynucleotide. The hairpin-linked fragments are contacted with a ligase, thereby ligating the hairpin-linked fragments together to form a single-stranded circular nucleic acid comprising a pair of opposing loops and an intervening duplex region which comprises a pair of nucleobase segment gaps. The single-single stranded circular nucleic acid is contacted with a polymerase and nucleotide triphosphates, thereby filling the nucleobase segment gaps.
In another aspect, a kit is provided for preparing single-stranded circular DNA. The kit comprises a hairpin polynucleotide comprising a transposase recognition sequence, a transposase, a polymerase and a ligase.
In another aspect, a system is provided for generating single-stranded circular nucleic acid from a target nucleic acid. The system comprises a first reaction chamber provided with a first set of reaction buffer components configured for formation of a complex between a transposase and a plurality of hairpin polynucleotides, each of the hairpin polynucleotides having a duplex region comprising a transposase recognition sequence. The system comprises a second reaction chamber provided with a second set of reaction buffer components configured for fragmenting the nucleic acid and ligating the hairpin polynucleotides to the nucleic acid, to form hairpin-linked nucleic acid fragments, each having a nucleobase segment gap between each fragment and its corresponding hairpin polynucleotide. The system further comprises a third reaction chamber provided with a third set of buffer components which is compatible with ligase, polymerase and nucleotide triphosphates. The system further comprises a liquid handler configured to transfer aliquots of solutions from the first reaction chamber to the second reaction chamber and from the second reaction chamber to the third reaction chamber.
In some embodiments of the system, a purification module is provided for purifying the single-stranded circular nucleic acid. The purification module is in liquid handling communication with the third chamber.
In some embodiments, the system is provided within a microfluidics chip.
In some embodiments, the methods further include the step of heating the single stranded circular nucleic acid to denature the intervening duplex region. Other method steps may further include mixing the hairpin-linked fragments with a kinase before or during the step of filling in the nucleobase segment gaps to phosphorylate any non-phosphorylated 5′-ends.
In certain embodiments of the methods and kits, the transposase is MuA transposase or Tn5 transposase. In embodiments where the transposase is Tn5 transposase, the transposase recognition sequence employed may be the 19-base pair mosaic end sequence of the Tn5 transposon. In other embodiments where the transposase is MuA transposase, the transposase recognition sequence used may be the R1 and/or the R2 region of the MuA transposon.
In some embodiments, the transposase catalyzes random integration of the hairpin polynucleotide into the target nucleic acid.
In some embodiments, the polymerase lacks 5′-3′ exonuclease activity and/or strand-displacement activity.
In some embodiments, the polymerase is T4 polymerase or T7 polymerase and the ligase is T4 ligase or E. coli ligase.
In some embodiments, the hairpin polynucleotides comprise sequencing tags.
In certain embodiments of the kits provided herein, instructions are provided for performing a series of reactions to produce single-stranded circular nucleic acid. The kit may be used to produce a library of single-stranded circular nucleic acids.
The single-stranded circular nucleic acid produced according to the method described herein can be used as a template for amplification or sequencing.
The library of single-stranded circular nucleic acids produced according to the method described herein can also be used for amplification or sequencing.
The foregoing summary and detailed description is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation.
The next generation of rapid nucleic acid sequencing technologies, such as single molecule real-time sequencing have the capability to re-sequence a single segment of double-stranded DNA repeatedly by using a single-stranded circular DNA molecule as a template (see for example, WO2009120372, Caruccio et al., Nextera™ Technology for NGS DNA Library Preparation: Simultaneous Fragmentation and Tagging by In Vitro Transposition Epicentre Forum 2009, 16-3, 4-6, which are incorporated herein by reference in entirety). Currently, DNA libraries comprising single-stranded circular DNA are produced using physical methods for fragmentation of DNA such as sonication and nebulization among others. This step is followed by selection of fragments of appropriate length and subjecting them to enzymatic processing to prepare the sample for sequencing. These physical methods require large amounts of DNA, on the order of microgram quantities, and are generally inefficient.
Methods are needed to improve the process for preparing single-stranded circular nucleic acids and libraries thereof in order to speed the “front-end” processing work required to take advantage of rapid sequencing technologies.
Transposons are found in all the biological kingdoms, and some perform specialized functions. For example, the genome of bacteriophage Mu includes a transposon that uses transposition both to integrate into the DNA of a new host cell and to replicate before lysis. Like most DNA rearrangements, transposition is a complex, multi-step process, requiring numerous DNA sequence elements. Studies of bacteriophage Mu have been central to our understanding of both the fundamental mechanisms and the complexities of DNA transposition.
Phage Mu encodes the MuA transposase which transfers the Mu genome from one DNA location (the transposition donor) to a new location (the transposition target). During transposition, transposase performs two principle reactions: DNA cleavage and DNA strand transfer. During cleavage, the donor DNA is nicked twice, once at each 3′-end of the Mu genome. During strand transfer, the cleaved transposon ends are inserted into neighboring sites on the two target strands.
Little or no specific sequence information is needed on the target DNA, but the Mu DNA provides many sequence cues for transposition. For example, the last two nucleotides at either 3′ end of the Mu DNA, the cleavage sites, have the sequence 5′-CA. Also near each end of the Mu DNA are three recognition sites, distinct from the cleavage sites, which share a 22-base pair consensus sequence. The recognition sites are referred to as R1, R2, and R3 on the right end and L1, L2, and L3 on the left end (
Another example of a transposase is Tn5 transposase, which has been adapted as a molecular biology reagent (EZ-Tn5™ Transposase) by Epicentre Biotechnologies Inc. (http://www.epibio.com). Applications of this reagent include in vitro insertion of an EZ-Tn5 Transposon into DNA cloned in vectors, such as plasmids, fosmids, cosmids, or BACs as well as in vitro insertion into linear DNA. The reagent may also be used for preparation of EZ-Tn5 transposomes for in vivo transposition following electroporation into living cells.
The EZ-Tn5™ Transposase reagent is a hyperactive form of Tn5 transposase. The highly purified, single-subunit enzyme can be used to randomly insert (transpose or “hop”) any EZ-Tn5 Transposon into any target DNA in vitro with an efficiency up to 106 insertion clones per standard reaction. When incubated with an EZ-Tn5 Transposon™ in the absence of Mg2+, a stable EZ-Tn5 Transposome™ complex is formed. The transposome is so stable that it can be electroporated into living cells. Once in the cell, the transposome is activated by intracellular Mg2+ and the EZ-Tn5 transposon component is randomly inserted into the host's genomic DNA.
A typical EZ-Tn5 transposition reaction requires four components: (1) the EZ-Tn5 Transposase™; (2) an EZ-Tn5 transposon; (3) a target DNA; and (4) the presence of Mg2+. The highly random insertion of an EZ-Tn5 transposon into the target DNA proceeds by a cut-and-paste mechanism catalyzed by the EZ-Tn5 Transposase™, and results in a 9-bp duplication of target DNA sequence immediately adjacent to both ends of the transposon.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Further, 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 to which this invention pertains. In describing and claiming the present invention, the following terminology and grammatical variants will be used in accordance with the definitions set forth below.
A “hairpin polynucleotide”, as used herein, is a single stranded polynucleotide having two regions which are sufficiently complementary that they hybridize to each other. Preferably, the two regions are completely complementary. As shown in
The nucleotides in the double stranded portion of the hairpin generally outnumber the nucleotides in the loop portion. For example, the double stranded portion of the hairpin can comprise about 60% or more of the nucleotides. In some applications, the double stranded portion of the hairpin can comprise more than about 90% of the nucleotides and, at times, more than about 99% of the nucleotides.
A hairpin polynucleotide that is not circular has a 3′ end and a 5′ end. The portion in the stem of the hairpin with the 3′ end is referred to as the “3′ portion.” The portion in the stem of the hairpin with the 5′ end is referred to as the “5′ portion.”
As used herein, the term “transposase” refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism.
As used herein, the term “transposon” refers to a sequence of nucleic acid which can move around to different positions within the genome of a single cell in a process known as transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons were also once called jumping genes, and are examples of mobile genetic elements. There are several mobile genetic elements and they can be grouped based on their mechanism of transposition. Class I mobile genetic elements, or retrotransposons, copy themselves by first being transcribed to RNA, then reverse transcribed back to DNA by reverse transcriptase, and then being inserted at another position in the genome. Class II mobile genetic elements move directly from one position to another using a transposase to “cut and paste” them within the genome.
As used herein, the term “ligase” refers to an enzyme which catalyzes the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group pendant to one of the larger molecules. For example, DNA ligase is an enzyme commonly used in molecular biology laboratories to join together DNA fragments. Other common names for ligases include synthetases, because they are used to synthesize new molecules.
As used herein, the term “polymerase” refers to an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA. The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription. In association with a cluster of other enzymes and proteins, they use free nucleotides (usually in the form of nucleotide triphosphates) in the solvent, and catalyze the synthesis of a polynucleotide sequence against a nucleotide template strand using base-pairing interactions.
As used herein, the terms “processivity” and “processive” refer to a measure of the average number of nucleotides added by a DNA polymerase enzyme per association/disassociation with the template. DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have low processivity. Multiple DNA polymerases have specialized roles in the DNA replication process. For example, in E. coli, which replicates its entire genome from a single replication fork, the polymerase DNA Pol III is the enzyme primarily responsible for DNA replication and forms a replication complex with extremely high processivity. The related DNA Pol I has exonuclease activity and serves to degrade the RNA primers used to initiate DNA synthesis. Pol I then synthesizes the short DNA fragments that were formerly hybridized to the RNA fragment. Thus Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions.
As used herein, the term “kinase” refers to an enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates. The process is referred to as phosphorylation. An alternative to the term “kinase” is “phosphotransferase.”
As used herein, the term “exonuclease” refers to an enzyme that cleaves nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction occurs that breaks phosphodiester bonds at either the 3′ or 5′ ends. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle of a polynucleotide chain. The related term “exonuclease activity” refers to the catalytic activity of an exonuclease.
As used herein, the term “strand-displacement activity” refers to the ability of a polymerase enzyme to displace a strand of an existing duplexed in the path of synthesis of a new strand. For example, in multiple-displacement amplification, the amplification reaction initiates when multiple primer hexamers anneal to the template. When DNA synthesis proceeds to the next starting site, the polymerase displaces the strand which was synthesized at that starting site and continues its strand elongation. For example, bacterial phage Φ29 DNA polymerase is a high proccessivity polymerase enzyme with strand displacement activity that can produce DNA 7 kb to 10 kb long. The reaction can be carried out at the moderate isothermal temperature condition of 30° C. It has been actively used in cell-free cloning, which is the enzymatic method of amplifying DNA in vitro without the need for cell culture and DNA extraction.
As used herein, the term “microfluidics” refers to technologies addressing the behavior, precise control and manipulation of fluids that are geometrically constrained to a sub-millimeter scale. It is a multidisciplinary field intersecting engineering, physics, chemistry, microtechnology and biotechnology, with practical applications to the design of systems in which such small volumes of fluids will be used.
As used herein, the terms “segment,” “fragment,” and “portion” when used in relation to polynucleotides or oligonucleotides of any kind, including the hairpin polynucleotides described herein, refer to a continuous sequence of nucleotide residues, which forms a subset of a larger sequence. For example, if a target nucleic acid is subjected to treatment with the transposase-hairpin polynucleotide complex, the oligonucleotides resulting from such treatment would represent segments or fragments of the starting target nucleic acid.
The hairpin polynucleotides used as starting materials in the methods described herein may be prepared synthetically, using either automation or conventional chemistry, for example by attaching a starting structure to beads and adding nucleotides thereto. Such methods are known to those skilled in the art. The hairpin oligonucleotides for use in the present invention may also be prepared by synthesizing segments or fragments thereof and then joining said segments or fragments into larger structures containing appropriate nucleotide sequences for use herein. Such segments or fragments may also be of natural origin, derived from microorganisms in nature or the result of cloning of sequences within selected organisms and utilizing selected vectors for the cloning process. Such segments or fragments may also be derived from natural vectors, such as plasmids, viruses, or the like.
The hairpin polynucleotides for use in the present invention may also be hybrids, or chimeras, containing some segments or sequences that are of natural origin as well as segments or sequences wholly synthetic in origin. The fact that a given segment or sequence is found in nature does not prevent it from being prepared synthetically in the laboratory for use herein.
An exemplary embodiment of a process of obtaining single-stranded circular nucleic acid from a double-stranded nucleic acid target is shown in
In the next step of the process (shown in the middle portion of
In the next step of the process (shown at the bottom of
In another embodiment, there is provided a system for producing single-stranded circular nucleic acid. The system may be provided at either typical laboratory scale or at microfluidics scale where components of the system are provided on a chip and under the control of a computer. The system is provided with a plurality of reaction chambers where different steps in the process are carried out. Segregation of the process steps is expected to be necessary because certain reagents, buffers and additives needed for certain steps may be incompatible with other steps. For example, Mg2+ is needed for polymerase reactions but is thought to interfere with formation of stable complexes of Tn5 transposase with hairpin polynucleotides. Therefore, a separate chamber for formation of the complex may be advantageous.
An example of this embodiment is shown in
The liquid handler 1002 is configured to obtain a sample S comprising target nucleic acid and transfer the sample S to the second chamber 1008. The liquid handler is also configured to transfer an aliquot of the solution containing the transposase-hairpin polynucleotide complex to the second chamber 1008. In some embodiments, it may be advantageous to provide a purification module, such as a reversed phase chromatography column (not shown) at the exit point of one or more of the process chambers 1006, 1008, 1010 for removing buffer additives, salts and reagents in the event that it is found that these components interfere with the function of the next process chamber. For example, if the buffer used for forming the complex in the first chamber 1006 is incompatible with the process of fragmenting target nucleic acid in the second chamber 1008 a purification module can be provided at the outlet of the first chamber 1006 where buffer exchange can be carried out according to established methods known to those skilled in the art.
Once the target nucleic acid has been fragmented by the transposase-hairpin polynucleotide complex, it is transferred by the liquid handler 1002 to the third chamber 1010 which is provided with the reagents and enzymes needed to ligate the fragments and fill in the nucleobase segment gaps. The end result is the production of single-stranded circular nucleic acid which is useful for rolling circle amplification and rapid sequencing methods.
In certain embodiments, the hairpin polynucleotides are provided with tags which are specific for various sequencing platform technologies. When subjected to the methods described above, these tagged hairpin polynucleotides produce tagged libraries. Examples of such tags have been described for producing Nextera and Roche/454-compatible libraries for rapid sequencing platforms (Caruccio et al., Nextera™ Technology for NGS DNA Library Preparation: Simultaneous Fragmentation and Tagging by In Vitro Transposition Epicentre Forum 2009, 16-3, 4-6., incorporated herein by reference in entirety).
In some embodiments, a single-stranded circular nucleic acid library representing the genome of an organism or a virus is used as a template for obtaining bioagent identifying amplicons which provide base compositions that provide the means for rapid identification of the organism or virus through mass spectrometry according to methods described in patents, patent applications and scientific publications, all of which are herein incorporated by reference as if fully set forth herein: U.S. Pat. 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While the present invention has been described with specificity in accordance with certain of its embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.
Hairpin polynucleotides of various lengths with varying stem lengths, stem loops, blunt ends and staggered ends are prepared using standard oligonucleotide synthesis methods known to those skilled in the art. The hairpin polynucleotides contain transposase recognition sequences such as the end mosaic sequence of Tn5 transposase or the R1 and R2 sequences of MuA transposase. The positions of the transposase recognition sequence within the duplex region of the hairpin polynucleotides may be varied with the objective of optimizing the molecular recognition of the sequence by the transposase. Hairpin polynucleotides can be prepared with platform-specific sequencing tags as well. In these cases, the tagged hairpin polynucleotides are tested to ensure that the tags do not appreciably interfere with the process of complex formation.
Complex formation may be monitored using gel electrophoresis, or other binding assay method such as surface plasmon resonance spectroscopy for example.
In selecting appropriate characteristics for candidate hairpin polynucleotides, it may be advantageous to undertake molecular modeling studies for binding of hairpin polynucleotides to the selected transposase using published three-dimensional structures of the transposons and the newly designed hairpin polynucleotides. Such molecular modeling methods are known to those with skill in the art.
Buffer conditions are optimized for enhancing the process of formation of the complex. For example, reagents or stabilizers, such as Mg2+ for example, which are known to interfere with the process of complex formation, are excluded from the buffer used for complex formation.
For development of methods described herein, a standard target nucleic acid, for example, a simple viral test genome is selected, such as the genome of H. influenzae which contains 1.8 million base pairs.
The transposome-hairpin polynucleotide complex optimized according to Example 1 is then incubated with the test genome for varying periods of time in order to prepare fragments of optimal sizes. The optimal sizes chosen may depend upon the ultimate application of the single-stranded circular nucleic acid produced. The incubation temperature is carefully controlled to control the activity level of the transposome complex.
Conditions which may be appropriate for the fragmentation reaction were described for MuA transposase (Goldhaber-Gordon et al., J. Biol. Chem. 2002, 277, 7694-7702, incorporated herein by reference in entirety) and may be adapted to the fragmentation reaction as follows. The reactions are conducted in a 25 μl volume containing 25 mM Tris-HCl (pH 8 at room temperature), 140 mM NaCl, 10 mM MgCl2Cl2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 15% glycerol, 12% dimethylsulfoxide, 0.1% Triton, 2 mM ATP, 250 ng of test DNA, and variable amounts of Mu DNA fragments and MuA. The MuA transposase is prepared by dilution of concentrated stock into 600 mM NaCl, 25 mM HEPES-KOH, 0.1 mM EDTA, 10% glycerol, and 1 mM dithiothreitol. The reactions are incubated at 30° C. for 20-60 min.
In this example, the fragments produced in Example 2 are treated with a ligase such as T4 ligase or E. coli ligase to ligate fragments together as indicated schematically in
The single-stranded circular nucleic acid obtained from these tests can be purified by known methods and analyzed by gel electrophoresis, mass spectrometry, or various spectroscopic methods known to those skilled in the art. The single-stranded circular nucleic acid may also be tested as a template for amplification reactions such as rolling circle amplification or in rapid sequencing methods according to established procedures known to those skilled in the art.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, Genbank accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety.
The present application claims priority to PCT application No. ______ filed Mar. 9, 2011 (filed concurrently herewith) and U.S. Provisional Application Ser. No. 61/312,332, filed Mar. 10, 2010, the entirety of each of which is herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61312332 | Mar 2010 | US |
