Patent Application
20140309118
The present disclosure relates to a method of preparing nucleic acid molecules, and more specifically to a method that enables efficient synthesis of long nucleic acid molecules.
Typically employed DNA synthesis procedures for scalable DNA construction have the following disadvantages: (a) high cost of oligonucleotides, (b) low assembly efficiency into long DNA sequences, (c) time-consuming cloning, and (d) high cost of target DNA sequence validation. Above all, the major synthesis costs are the costs of oligonucleotides and sequencing. It would thus be desirable to design a protocol for massively parallelizing synthesis products in order to achieve effective, highly scalable DNA synthesis. DNA oligonucleotides derived from DNA microchips have previously been utilized to synthesize scalable low-cost DNA (Tian, J., et al., 2004). However, the low assembly efficiency of chip-derived oligonucleotides hinders target gene construction, and a laborious DNA assembly optimization process is consequently required. The inefficiency of DNA assembly from chip-derived oligonucleotides is largely associated with the incomplete removal of flanking regions of double-stranded (ds)-oligonucleotides prior to their assembly and the uneven concentration of each chip-cleaved oligonucleotide (Kim H., et al., 2011). Furthermore, it was observed that a greater number of oligonucleotides (i.e. higher complexity) in a DNA assembly pool made DNA assembly less efficient (Kim H., et al., 2011; Borovkov A. Y., et al., 2010). As a consequence, only a small sub-pool of oligonucleotides (i.e. <20) are often amplified to ensure high assembly efficiency. There is a need to develop a high-efficiency DNA assembly process using a large number of microchip oligonucleotides present in a pool in order to attain all advantages of ultra-low cost DNA microchip oligonucleotides.
For scalable DNA synthesis, it is preferable to decrease the sequencing cost for target DNA validation. In recent years, costs for high-throughput sequencing technologies have been considerably lowered. Under such circumstances, utilization of high-throughput sequencing technology has great potential for DNA synthesis at ultra-low cost. However, unlike colony-based Sanger sequencing validation, it is difficult to collect the desired DNA from a pool of high-throughput sequenced DNA mixtures. Although recent high-throughput sequencing technologies can be applied to partially addressable spots (for example, clonal spots available from Roche-454, Illumina and SOLiD, and single-molecule spots available from Helicos and PacBio), it is difficult to isolate target DNA due to the difficulty associated with the collection of the desired DNA from high-throughput sequencing plates. In a notable report (Matzas M., et al., 2010), chip-cleaved oligonucleotides were sequenced by 454 sequencing technology, and directly isolated from the 454 sequencing plate using a bead picker pipette. These sequence-validated ‘oligonucleotides’ were subsequently processed and used to assemble 200 bp target DNA fragments. This study demonstrates the possibility of convergence of next-generation sequencing technology and microchip oligonucleotides in terms of DNA synthesis cost reduction. In this study, however, high-throughput sequencing was carried out on chip oligonucleotides rather than on assembled DNA fragments. Accordingly, challenges associated with DNA assembly into larger sequences are still in early stages. Furthermore, an effective error-free oligonucleotide picking process necessitates a highly tuned bead picking robot and an image processing system.
A number of papers and patent publications are referenced and cited throughout the specification. The disclosures of the papers and patent publications are incorporated herein by reference in their entireties in order to more fully describe the state of the art to which the present disclosure pertains and the disclosure of the present disclosure.
According to one embodiment of the present disclosure, there is provided a method of preparing nucleic acid molecules, including (a) providing nucleic acid fragments constituting at least a portion of the complete sequence of a target nucleic acid, (b) tagging the nucleic acid fragments with barcode sequences, (c) validating the sequences of the nucleic acid fragments tagged with the barcode sequences, and (d) recovering desired nucleic acid fragments among the sequence-validated nucleic acid fragments using the barcode sequences.
According to a further embodiment of the present disclosure, there is provided a method of preparing nucleic acid molecules, including (a) providing nucleic acid fragments constituting at least a portion of the complete sequence of a target nucleic acid, (b) assembling the nucleic acid fragments to synthesize intermediates having sizes whose sequences are validatable by a parallel sequencing technology, (c) tagging the intermediates with barcode sequences, (d) validating the sequences of the intermediates tagged with the barcode sequences, (e) recovering desired intermediates among the sequence-validated intermediates using the barcode sequences, and (f) assembling the recovered intermediates to form long nucleic acid molecules.
According to another embodiment of the present disclosure, there is provided a method of preparing nucleic acid molecules, including (a) providing a pool of oligonucleotides containing restriction enzyme digestion sequences and generic flanking sequences, (b) cleaving the restriction enzyme digestion sequence portions to provide a pool of mixtures including the oligonucleotides, each containing the generic flanking sequences at one end, and the oligonucleotides, each containing none of the generic flanking sequences at one end, and (c) assembling the oligonucleotides using the generic flanking sequences to randomly synthesize nucleic acid fragments.
According to yet another embodiment of the present disclosure, a method of preparing nucleic acid molecules, including (a) providing a pool of oligonucleotides, (b) assembling the oligonucleotides to randomly synthesize nucleic acid fragments, (c) connecting base sequences for amplification to the randomly synthesized nucleic acid fragments, and (d) amplifying the nucleic acid fragments with primers bound to the base sequences for amplification.
Embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. These embodiments are provided so that this disclosure will fully convey the scope of the disclosure to those skilled in the art. Accordingly, the present disclosure may be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. In the drawings, the dimensions, such as widths, lengths and thicknesses, of elements may be exaggerated for convenience. It will be understood that when a first element is referred to as being “connected” or “attached” to a second element, the first element can be directly connected or attached to the second element or a third element may also be interposed between the first and second elements.
When it is intended to synthesize large target nucleic acid molecules, the nucleic acid fragments may be ones that are free of sequence errors such as insertion, deletion, transition and transversion.
The nucleic acid fragments provided in step S110 may be directly extracted from a pool of oligonucleotides. Alternatively, the nucleic acid fragments may be prepared by amplifying and assembling oligonucleotides so as to have lengths above a predetermined level. When it is intended to synthesize long target nucleic acid molecules, the nucleic acid fragments may be made by various processes, including a hierarchical gene synthesis process (Journal of Biotechnology 151 (2011) 319-324) or a random gene synthesis process, which will be described below.
In the present specification, random gene synthesis is also referred to as “shotgun synthesis”, and nucleic acid fragments made by such a shotgun synthesis method are also referred to as “shotgun products.”
Shotgun sequencing is a process in which analyte DNA is randomly fragmented, sequencing adaptors are connected to the nucleic acid fragments, followed by high-throughput sequencing analysis. Shotgun sequencing includes arranging the individual fragments and identifying the complete sequence of the original analyte DNA using a computer program. Shotgun synthesis proceeds in the exact reverse order to that of the shotgun sequencing. Oligonucleotides constituting a portion of the sequence of nucleic acid molecules to be synthesized are constructed and assembled randomly to make nucleic acid fragments, which are analyzed by high-throughput sequencing. Desired nucleic acid fragments are recovered among the analyzed nucleic acid fragments and are used to make the final nucleic acid molecules.
According to one embodiment of the present disclosure, the nucleic acid fragments provided in step S110 may be shotgun products prepared by a shotgun synthesis method. Oligonucleotides designed to contain generic flanking sequences may be used to make the shotgun products.
In step S210, the generic flanking sequence may exist at one or both ends of the oligonucleotide. For example, the oligonucleotides used in the random gene synthesis (shotgun synthesis) process may contain, from the 5′ to 3′ direction, 5′-end generic flanking sequences, the oligonucleotide sequences constituting the target nucleic acid, and 3′-end generic flanking sequences.
The 5′-end generic flanking sequences and 3′-end generic flanking sequences existing at the ends of the oligonucleotides are priming regions where the amount of the oligonucleotide derived from DNA chips is amplified, and are used as annealing regions of primer sets for the production of a sufficient amount of the oligonucleotides.
The oligonucleotides may contain restriction enzyme digestion sequences. The nucleic acid fragments contain 5′-restriction enzyme digestion sequences with the 5′-end generic flanking sequences, and 3′-restriction enzyme digestion sequences with the 3′-end generic flanking sequences. The 5′-restriction enzyme digestion sequences and the 3′-restriction enzyme digestion sequences in the oligonucleotides may be identical to or different from each other.
The oligonucleotides are 50-500 base pairs (bp), more preferably 100-300 bp, even more preferably 120-200 bp, most preferably about 150 bp in length.
According to one embodiment of the present disclosure, the oligonucleotides may contain portions or all of the sequence of the target nucleic acid. When the oligonucleotides contain portions of the sequence of the target nucleic acid, the target oligonucleotides with varying sizes are sequentially assembled to synthesize the target nucleic acid molecules containing all of the sequence.
The pool of the oligonucleotides may be one that is cleaved from DNA microchips. Alternatively, the pool of the oligonucleotides may be a mixture of oligonucleotides synthesized on a solid. The cleaved oligonucleotides may be amplified to ensure an amount necessary for long gene synthesis. This amplification may be perform by polymerase chain reaction (PCR) using the generic flanking sequences.
Next, the generic flanking sequences are cleaved using a restriction enzyme recognizing the restriction enzyme digestion sequences in the amplified oligonucleotides. The pool of the cleaved oligonucleotides may take the form of a mixture including the oligonucleotides, each containing none of the generic flanking sequences because the restriction enzyme digestion sequences at both ends are completely cleaved, and the oligonucleotides, each containing the generic flanking sequences remaining at one end because only the restriction enzyme digestion sequences at one end are cleaved.
The oligonucleotides of the mixtures can be assembled by polymerase chain reaction assembly (PCA) using the generic flanking sequences. At this time, the oligonucleotides are sequentially assembled to make fragments with varying lengths. Such fragments may be randomly assembled to each other. Thus, the small or large fragments may be randomly assembled at various locations in the PCR solution to synthesize longer fragments containing all or portions of the sequence of the target nucleic acid molecules. This assembly may proceed until the oligonucleotides, each containing the generic flanking sequence at one end, overlap each other to make nucleic acid fragments containing the generic flanking sequences at both ends.
The oligonucleotides of step S210 are elaborately designed to form desired shotgun products. Several oligonucleotides may be assembled in such a manner that they overlap each other through some complementary sequences of the oligonucleotide sequences. The oligonucleotides are designed for random assembly to form shotgun products. For example, if a shotgun product (e.g., ˜400 bp) containing the 5′-end regions of the target nucleic acid molecules consists of 5 target oligonucleotides, it may be formed through sequential assembly among the following oligonucleotides cleaved using restriction enzymes: from the 5′ to 3′ direction, to form a 5′-end region, a first oligonucleotide containing a 5′-end generic flanking sequence and a portion of the sequence of the target nucleic acid molecules and from which the restriction enzyme digestion sequences are partially cleaved; a second oligonucleotide including a region (e.g., 20-50 bp long) overlapping the 3′-end region of the first oligonucleotide; a third oligonucleotide including a region overlapping the 3′-end region of the second oligonucleotide; a fourth oligonucleotide including a region overlapping the 3′-end region of the third oligonucleotide; and a fifth oligonucleotide containing a sequence including a region overlapping the 3′-end region of the fourth oligonucleotide and a 3′-end generic flanking sequence.
In a modified embodiment, the nucleic acid fragments may be prepared by the following method.
First, a pool of oligonucleotides is provided. Next, raw oligonucleotides without the addition of generic flanking sequences, etc. are assembled to randomly synthesize nucleic acid fragments, unlike the previous embodiment. Base sequences for amplification are connected to the randomly synthesized nucleic acid fragment, and then the nucleic acid fragments are amplified with primers bound to the base sequences for amplification to obtain amplified nucleic acid fragments.
As described above, the preparation of nucleic acid molecules by random synthesis processes is advantageous in that several kinds of libraries of nucleic acid fragments can be prepared simultaneously.
According to one embodiment of the present disclosure, the nucleic acid fragments of step S110 may include the complete sequence of a target nucleic acid. For the synthesis of error-free long DNA, the sequences of the nucleic acid fragments may be validated using a parallel sequencing system. When the performance of the parallel sequencing system to validate the sequences of the nucleic acid fragments is taken into consideration, the nucleic acid fragments are preferably 20-3,000 bp, more preferably 200-1,000 bp, more preferably 300-500 bp, even more preferably 350-450 bp, most preferably 380-420 bp in length. Despite this preferred numerical range, an improvement in the performance of parallel sequencing systems for the analysis of several thousand by long DNA can extend the size of the nucleic acid fragments to several thousand by long DNA.
The term “nucleotide” as used herein refers to a single- or double-stranded deoxyribonucleotide or ribonucleotide and includes naturally occurring nucleotide analogs unless stated otherwise (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
The term “oligonucleotide” as used herein refers to an oligomer or polymer of nucleotides or an analog thereof. According to one embodiment of the present disclosure, the gene amplification is carried out by PCR. According to one embodiment of the present disclosure, the primers (for example, the generic flanking sequences) are used in gene amplification reactions.
The term “amplification reactions” as used herein refers to reactions for amplifying target nucleic acid sequences. Various amplification reactions were reported in the art and include, but are not limited to, polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), the methods of Miller, H. I. (WO 89/06700) and Davey, C. et al. (EP 329,822), multiplex PCR (McPherson and Moller, 2000), ligase chain reaction (LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) (WO88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (APPCR) (U.S. Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA) (U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), and strand displacement amplification (21, 22). Other possible amplification methods are described in U.S. Pat. Nos. 5,242,794, 5,494,810, and 4,988,617, and U.S. patent application Ser. No. 09/854,317.
In a most preferred embodiment of the present disclosure, the amplification procedure is carried out in accordance with PCR disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.
PCR is one of the most well-known nucleic acid amplification methods and many modifications and applications thereof have been developed. For example, traditional PCR procedures have been modified to develop touchdown PCR, hot start PCR, nested PCR, and booster PCR with improved PCR specificity or sensitivity. In addition, multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. Details of PCR can be found in McPherson, M. J., and Moller, S. G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference. Examples of preferred target nucleic acid molecules that can be used in the present disclosure include, but are not particularly limited to, DNA (gDNA and cDNA) and RNA. DNA is more preferred. Examples of target nucleic acids suitable for use in the present disclosure include nucleic acids from prokaryotic cells, eukaryotic cells (e.g., protozoans, parasites, bacteria, yeasts, higher plants, lower animals, and higher animals, including mammals and humans), viruses (e.g., herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, and poliovirus), and viroids.
The primers used in the present disclosure are hybridized or annealed to sites of the template to form double-stranded structures. Suitable conditions of nucleic acid hybridization for the formation of such double stranded structures are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).
A variety of DNA polymerases can be used for amplification in the present disclosure and include “Klenow” fragment of E. coli DNA polymerase I, thermostable DNA polymerases, and bacteriophage T7 DNA polymerase. Preferred are thermostable DNA polymerases that can be obtained from a variety of bacterial species, including DNA polymerases and Phusion polymerases of Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Pyrococcus furiosus (Pfu), Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana and Thermosipho africanus. Most preferably, Pyrococcus furiosus (Pfu) or Phusion high-fidelity DNA polymerase is used.
When the polymerization reaction is carried out, it is preferred to provide excessive amounts of the components necessary for amplification to a reaction vessel. The excessive amounts of the components necessary for amplification refer to amounts of the components in which the amplification reaction is not substantially limited by the concentrations of the components. It is desirable to provide, to the reaction mixture, cofactors such as Mg2+ and dATP, dCTP, dGTP and dTTP in amounts sufficient to reach a desired degree of amplification. All enzymes used in the amplification reaction may be active under the same reaction conditions. Indeed, a buffer allows all enzymes to reach their optimum reaction conditions. Thus, the use of a buffer enables the amplification of a single reactant without any change in reaction conditions such as the addition of other reactants.
In the present disclosure, annealing is carried out under stringent conditions that allow for specific binding between the target nucleotide sequences (e.g., the generic flanking sequences of the target oligonucleotides) and the primers. The stringent annealing conditions are sequence-dependent and vary depending on ambient environmental parameters. The oligonucleotide pool thus amplified can be used to make primary amplification products. The primary amplification products can be used to prepare secondary amplification products, which can be assembled into larger target nucleic acid molecules (e.g., ≧10 kb).
The term “primer” as used herein refers to an oligonucleotide that can act as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand (a template) is induced, i.e., in the presence of nucleotides and a polymerase, such as DNA polymerase, and under appropriate temperature and pH conditions. Preferably, the primer is deoxyribonucleotide and a single strand. The primers used in the present disclosure may include naturally occurring dNMP (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides, and non-naturally occurring nucleotides. Other examples of the primers include ribonucleotides.
The primers should be sufficiently long to prime the synthesis of extension products in the presence of a polymerase (such as DNA polymerase). The length of the primers may vary depending on many factors, e.g., temperature, application, and sources of the primers. The primers are typically 15-30 nucleotides long. Short primer molecules generally necessitate a lower temperature to form sufficiently stable hybridization composites with templates.
The term “annealing” or “priming” as used herein refers to the apposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid. The apposition enables the polymerase to polymerize nucleotides into a nucleic acid molecule complementary to the template nucleic acid or a portion thereof. The term “hybridization” as used herein refers to a process in which two single-stranded nucleic acids form a duplex structure by pairing of complementary base sequences. The hybridization may occur when complementarity between single-stranded nucleic acid sequences is perfectly matched or even when partially mismatching bases are present. The degree of complementarity necessary for hybridization may vary depending on hybridization reaction conditions, particularly temperature.
The term “complementary” as used herein means a level of complementarity sufficient to selectively hybridize with the nucleotide sequence under certain particular hybridization or annealing conditions, and is intended to include both substantially complementary and perfectly complementary, preferably perfectly complementary.
Referring back to
The kinds of the barcode sequences are not particularly limited so long as they can be added to distinguish the nucleic acid fragments from each other. The number of the kinds of the barcode sequences is preferably greater than that of the nucleic acid fragments to distinguish the individual nucleic acid fragments. For example, the barcode sequences may be mixtures of two or more kinds of randomly or intentionally designed oligonucleotides.
According to one embodiment of the present disclosure, poly-N degenerate-barcode sequences among the barcode sequences may use poly-N degenerate DNA or may also use sequences barcoded with two or more different sequences randomly made using a computer program well known in the art.
The tagging with the barcode sequences is not particularly limited and may be performed by a method selected from the group consisting of PCR, emulsion PCR and ligation. For example, assembly of the barcode sequences to shotgun synthesized DNA fragments by PCR or ligation of double-stranded (ds) DNA including poly-N degenerate-barcode sequences may be used for the tagging.
In step S130, the sequences of the nucleic acid fragments tagged with the barcode sequences are validated. Parallel sequencing is preferably used to validate the sequences of the tagged nucleic acid fragments. As a result, the sequences of the tagged nucleic acid fragments, together with the tagging barcode sequences, can be validated.
According to one embodiment of the present disclosure, the parallel sequencing or high-throughput sequencing is carried out by a suitable method well known in the art, for example, using a Roche-454 sequencing system or a high-throughput sequencing system with a read length of 100 bp or more.
According to one embodiment of the present disclosure, sequencing adaptor sequences may be further added to the barcode sequences. Sequences containing the barcode sequences added to the nucleic acid fragments are herein referred to as “barcode primers” for convenience.
The term “adaptor sequences” as used herein refers to sequences that enable high-throughput sequencing analysis of the nucleic acid fragments. For example, the adaptor sequences includes all commercially available sequences for 454-sequencing used in the present disclosure. Examples of preferred adaptor sequences include, but are not limited to, adaptor sequences of Roche-454 sequencing platforms and adaptor sequences of other next-generation sequencing technologies.
The term “generic flanking sequences” as used herein refers to base sequences that are added to both ends of the oligonucleotides to selectively amplify particular oligonucleotides among the pool of oligonucleotides. The base sequences added to the 5′-ends of different oligonucleotides necessary for assembly into target nucleic acid molecules are identical to each other, and the base sequences added to the 3′-ends of different oligonucleotides are identical to each other.
According to one embodiment of the present disclosure, an amplification procedure using the primers bound to the adaptor sequences may be performed using the tagged nucleic acid fragments as templates for sequence validation.
The barcode sequences are not limited to particular lengths and are, for example, 5-300 bp, preferably 10-100 bp, more preferably 12-40 bp, even more preferably 15-30 bp in length taking into consideration the sequencing performance on the entire sequences including the nucleic acid fragments. This numerical range may vary with the advance of sequencing technologies. For example, when the poly-N degenerate-barcode sequences are 20 bp long, 420 kinds of the barcode sequences are possible.
The barcode primers may contain, for example, from the 5′ to 3′ direction, 454-adaptor sequences, poly-N degenerate-barcode sequences, restriction enzyme digestion sequences, and generic flanking sequences. The primers for amplification may be designed to bind to the 454-adaptor sequences.
The sequence validation enables identification of error-free nucleic acid fragments among the nucleic acid fragments and the barcode sequences added thereto.
On the other hand, the restriction enzyme digestion sequences contained in the barcode primers serve to remove the sequencing adaptor sequences of the nucleic acid fragments. The reason for this removal is because the presence of the adaptor sequences hinders subsequent assembly of the nucleic acid fragments because of attached beads in sequencing analysis.
In step S140, desired nucleic acid fragments are recovered among the sequence-validated nucleic acid fragments using the barcode sequences. The validation of the sequences of the desired nucleic acid fragments and the tagging barcode sequences by sequencing in the previous step enables recovery of the desired nucleic acid fragments using the barcode sequences. Specifically, the recovery step may be carried out by selectively amplifying the desired nucleic acid fragments with primers corresponding to the barcode sequences and recovering the amplified nucleic acid fragments. Alternatively, the recovery step may be carried out by selectively hybridizing the desired nucleic acid fragments with oligonucleotides corresponding to the barcode sequences and recovering the hybridized nucleic acid fragments. For example, the desired nucleic acid fragments may be error-free nucleic acid fragments.
The desired nucleic acid fragments may be recovered using a computer program. Specifically, the sequences of the nucleic acid fragments are imaginarily assembled using a computer program and are compared with the complete sequence of desired target nucleic acid molecules. Thereafter, primers synthesized based on the most optimized information on sequences flanking DNA fragments or primers hybridizing therewith can be used to recover the desired nucleic acid fragments.
According to one embodiment of the present disclosure, the computer program may be any of those known in the art. Examples of more preferred computer programs include in-house Python programs and programs constructed using Perl, C, C++ or other programming languages.
According to one embodiment of the present disclosure, the computer program is used to synthesize sequences complementary to the selected barcode sequences into oligos. Next, only error-free fragments capable of optimizing the synthesis of target DNA are recovered among the nucleic acid fragments (i.e. mixtures of erroneous fragments and error-free fragments) by amplification (PCR) or hybridization using the synthesized barcode oligos. Examples of methods for the recovery of error-free fragments using the synthesized barcode sequences include, but are not limited to, DNA capture methods using microchips and hybridization methods for recovering desired error-free fragments by attaching desired barcode sequences to biotinylated beads or magnetic beads, in addition to PCR.
According to one embodiment of the present disclosure, when the nucleic acid fragments are provided by shotgun assembly, the length of the error-free barcoded nucleic acid fragments may be 200 bp or more. When a next-generation sequencing system capable of analyzing DNA with 1,000 bp or more is used, the error-free barcoded nucleic acid fragments may be 1,000 bp or more in length. More preferably, the error-free barcoded nucleic acid fragments are from about 200 bp to about 10 kb or more in length.
In step S150, recovered nucleic acid fragments can be assembled to form long nucleic acid molecules.
According to one embodiment of the present disclosure, the target nucleic acid molecules prepared by the present disclosure include, but are not limited to, target genes, target gene clusters, target genomes, and natural or synthetic nucleic acid molecules.
The term “target gene cluster” or “target genome” as used herein refers to a cluster or genome that includes at least two genes encoding a desired target (gene). The cluster or genome may include cluster or genome regions capable of generating two or more gene products (e.g., genome regions including one or more multiple splicing products of the same gene).
According to one embodiment of the present disclosure, a target gene cluster or target genome that can be synthesized by the method of the present disclosure may have a length of about 10 kb or longer. For example, the target gene cluster or target genome may include a penicillin biosynthetic gene cluster DNA sequence (11,376 bp) from Penicillium chrysogenum, and the penicillin biosynthetic gene cluster may include pcbAB, pcbC, and penDE genes.
The term “natural or synthetic nucleic acid molecules” as used herein is intended to include DNA (gDNA and cDNA) and RNA molecules, and nucleotides as basic units of the nucleic acid molecules include not only natural nucleotides but also analogues having modified sugar or base moieties (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
Nucleic acid fragments constituting at least a portion of the complete sequence of a target nucleic acid are provided (step (a)). The size of the nucleic acid fragments provided in step (a) may be from 20 to 300 bp.
The nucleic acid fragments are assembled to synthesize intermediates having sizes whose sequences can be validated by a parallel sequencing technology (step (b)). The size of the intermediates is not particularly limited and may be, for example, from 50 to 3,000 bp. The intermediates may be increased to a desired size with the advance of parallel sequencing technologies such as next-generation sequencing technology. The intermediates may be synthesized by various synthesis processes, including hierarchical synthesis or random synthesis (shotgun synthesis).
The intermediates are tagged with barcode sequences (step (c)). Preferably, sequencing adaptor sequences are added to the barcode sequences for sequence validation.
The sequences of the intermediates tagged with the barcode sequences are validated (step (d)). The sequence validation of the intermediates tagged in step (d) may be performed by a parallel sequencing technology. The method may further include amplifying the tagged nucleic acid fragments using the sequencing adaptor sequences between steps (c) and (d).
Desired intermediates are recovered among the sequence-validated intermediates using the barcode sequences (step (e)). The desired intermediates may have error-free sequences.
The recovered intermediates are assembled to form long nucleic acid molecules (step (f)). The size of the long nucleic acid molecules may be 1,000 bp or more.
Referring to
h shows three bands obtained by excising the bands shown in
The foregoing embodiments of the present disclosure offer the following advantages.
The method of the present disclosure enables scalable synthesis of large target nucleic acid molecules in a more economical and efficient manner. According to the method of the present disclosure, amplification products containing the sequence of a target nucleic acid are prepared using an elaborately designed target oligonucleotide pool, 300-500 bp error-free shotgun assembly fragments are selectively recovered from the amplification products using barcode sequences, and larger target nucleic acid molecules (e.g., ≧˜10 kb) are synthesized using the error-free shotgun assembly fragments. In addition, the method of the present disclosure enables gene synthesis at lower cost than conventional methods using resin-based oligonucleotides. Therefore, the present disclosure can be applied as a novel method for the synthesis of large target nucleic acid molecules and thus provides very excellent means that can considerably reduce gene synthesis cost.
The present disclosure will be explained in more detail with reference to the following examples. These examples are provided for illustrative purposes only and it will be obvious to those skilled in the art that are not intended to limit the scope of the present disclosure in accordance with the subject matter of the present disclosure.
AccuPrep™ gel purification kits for DNA purification and AccuPrep™ plasmid extraction kits for plasmid extraction were purchased from Bioneer (Korea). Pfu polymerase pre-mix and Taq polymerase pre-mix were purchased from Solgent (Korea). Phusion polymerase pre-mix, restriction enzymes [EarI (20,000 U/ml) and BtsI (10,000 U/ml)], NEB buffer 4(10) and competent cells (C-2566) were purchased from New England Biolabs (NEB) (USA). TOP Cloner™ Blunt core kits (6 TOP cloner buffer, sterile water, pTop blunt V2) were purchased from Enzynomics (Korea). Microchip oligonucleotides and primers were purchased from Agilent (USA) and Macrogen (Korea), respectively. Sanger sequencing and Roche-454 sequencing were requested to Macrogene (Korea).
Target Penicillin Biosynthetic Gene Cluster and Oligonucleotide Sequence Design
Penicillin biosynthetic gene cluster (N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase) DNA sequence (11,376 bp) from Penicillium chrysogenum was chosen as a synthetic model. A codon-optimized penicillin biosynthetic gene cluster sequence was designed using the web-based program Optimizer (Puigb, P. et al., 2007). Twenty-four nucleotides (5-GCAGAGTAAAGACCGTGCACTTAT-3 SEQ ID NO: 1) were added to the microchip oligonucleotides.
Each Agilent chip oligonucleotide was 150 nucleotides in length and consisted of flanking sequences and target DNA sequences. Oligonucleotides (114 plus and 114 minus strands) for target DNA sequences were designed in such a way that upon annealing, complementary oligonucleotides contained overlapping regions for assembly. These 228 oligonucleotide sequences were flanked by generic PCR primer sequences.
Processing of Sub-Pools of Agilent Microchip Oligonucleotides
Lyophilized Agilent microchip oligonucleotides were suspended in 100 μl water. A higher concentration of the microchip oligonucleotide subpool (228 oligonucleotides targeting the penicillin biosynthetic gene cluster) was prepared using PCR amplification with flanking primers. The components included in each PCR reaction mixture were 8 μl water, 10 μl 2 Pfu polymerase pre-mix, 0.5 μl cleaved oligonucleotide pool, and 1 μl 10 μM forward and reverse primers. The first PCR reaction was performed as follows: (a) a pre-denaturation step at 95° C. for 3 min; (b) a 20-cycle PCR step, each cycle consisting of 95° C. for 30 s, 55° C. for 30 s, and 72° C. for 1 min; and (c) a final elongation step at 72° C. for 10 min. Thereafter, to amplify the oligos, the second PCR reaction was performed on the PCR products amplified by the first PCR reaction. For the PCR, the following reagents were used: 18 μl water, 25 μl 2 Pfu polymerase pre-mix, 3 μl of the first PCR products, and 2 μl 10 μM forward and reverse primers. The second PCR conditions were the same as for the first PCR reaction with the exception of the number of reaction cycles (i.e. 12). After verification of the desired products by 4% agarose gel electrophoresis, restriction enzyme digestion was carried out as follows: when EarI was used, 2.5 μl EarI, 5 μl NEB buffer, 0.5 μl 100×BSA and 50 μl PCR products were mixed, followed by digestion at 37° C. for 3 h; and when BtsI was used, 2.5 μl BtsI, 5 μl NEB buffer, 0.5 μl 100×BSA and 50 μl PCR products were mixed, followed by digestion at 55° C. for 3 h. The restriction digest products were electrophoresed through 4% agarose gels and gel-purified.
Shotgun Assembly
The gel-purified products were assembled using the first round shotgun assembly PCR. For the PCR, the following reagents were used: 20 μl Pfu polymerase pre-mix and 20 μl purified products (the sub-pool of 228 microchip oligonucleotides). The PCR conditions were as follows: a pre-denaturation step at 95° C. for 3 min; (b) a 36-cycle PCR step, each cycle consisting of 95° C. for 30 s, 60° C. for 30 s, and 72° C. for 1 min; and (c) a final elongation step at 72° C. for 10 min. After the PCR products were electrophoresed through an agarose gel (1.5%), gel regions (target size=˜350 bp) of 300-500 bp were excised.
Processing of the Shotgun Assembly Products by Barcoding and 454 Sequencing
The detailed procedure is illustrated in
The fragments were barcoded by a primer pair that consisted of, from the 5′ to 3′ direction, a 454 DNA sequencing-adaptor sequence, a 454 high-throughput sequencing key sequence (e.g., 5-TCAG-3), a 20-mer degenerate primer (i.e. made of poly N sites), an EcoP15I Type IIS restriction enzyme site, and the flanking primer sequences. The EarI or BtsI site was located at the 3′ end of the flanking sequence of the chip oligonucleotides. The EcoP15I site was introduced into the PCR amplification procedure for shotgun assembly of the fragments using the barcoded primers. For the PCR, the following reagents were used: 6 μl water, 20 μl 2 Pfu polymerase pre-mix, 10 μl the assembled gene fragment pool, and 2 μl forward and reverse barcode primers. The PCR conditions were as follows: (a) a pre-denaturation step at 95° C. for 3 min; (b) a 18-cycle PCR step, each cycle consisting of 95° C. for 30 s, 55° C. for 30 s, and 72° C. for 1 min; and (c) a final elongation step at 72° C. for 10 min. After the PCR products were electrophoresed through an agarose gel (1.5%), the gel was excised to purify assembled fragments (450-600 bp). These gel-purified fragments were diluted 100-fold and the diluted products were then used for a final PCR amplification step involving 454 DNA sequencing-adaptor primers (Macrogene, Korea). For the PCR, the following reagents were used: 17.5 μl water, 25 μl Pfu, 2.5 μl of the 100-fold diluted gel-purified products and 2.5 μl forward and reverse primers. Eight replicate 20 μl PCR reaction products. The PCR reaction conditions were as follows: (a) a pre-denaturation step at 95° C. for 3 min; (b) a 25-cycle PCR step, each cycle consisting of 95° C. for 30 s, 71° C. for 30 s, and 72° C. for 1 min; and (c) a final elongation step at 72° C. for 10 min. Thereafter, the PCR products were electrophoresed through an agarose gel (1.5%), followed by gel purification (450-500 bp). The eight replicates were pooled prior to 454 sequencing.
Prior to 454 sequencing, cloning of the barcoded target gene fragments was performed, and several colonies were selected and submitted for Sanger sequencing evaluation. Gel-purified and barcoded products were cloned into the TOPO vector using the TOP Cloner™ Blunt core kit (Enzynomics, Korea). Competent cells derived from C2566 (New England Biolabs, USA), an Escherichia coli strain, were then transformed with the cloned products. After overnight growth on agar plates at 37° C., several colonies were chosen for colony PCR using M13F-pUC and M13R-pUC universal primer pairs. After confirmation of the presence of inserted DNA, Sanger sequencing was conducted prior to Roche-454 sequencing. Thereafter, the sequences of the gene fragments and the barcode primer sequences were validated using the Lasergene program (DNAstar, Madison, Wis.). After verification of the sequences, the pool of assembly PCR products was selected for Roche-454 high-throughput sequencing. The sequencing data were analyzed using an in-house Python program, and error-free gene fragments were selected.
Algorithm of In-House Python Program to Analyze the 454 High-Throughput Sequencing
The primary task of the computer program was to select error-free shotgun assembly samples for subsequent assembly. The 454 sequencing read results (454 reads) were aligned to the target penicillin biosynthetic gene cluster sequence using the in-house Python programming language. DNA fragments with desired restriction enzyme sites (i.e. EcoP15I and either, EarI or BtsI sites) at both ends of the read were selected based on the sequencing data with a high quality score (Phred-like consensus quality >30, which corresponded to a base call accuracy >99.9%). Flanking sequences containing the enzyme site were eliminated from the processed gene fragments, and the flanking sequence-removed internal sequences were aligned to the target penicillin biosynthetic gene cluster sequence. When these internal sequences matched perfectly with the reference sequence, the aligned sequences were graphically listed along with their target gene cluster sequence (
These selected gene fragments were recombined into the complete target gene (
Synthesis of the Target Gene Cluster from the Target Assembly Products
Amplification of the Desired Shotgun Assembly Products and Elimination of the Flanking Sequences from the Shotgun Assembly Products
As described above, an in-house Python program was used to select optimum sets of shotgun assembly products. These overlapping error-free DNA fragments were selectively amplified from shotgun assembly DNA mixtures using selected barcode primer pairs. For the PCR, the following reagents were used: 8 μl water, 10 μl Phusion polymerase pre-mix, 1 μl forward and reverse barcode primers, and 1 μl of the shotgun assembly DNA mixture.
The PCR conditions were as follows: (a) a pre-denaturation step at 95° C. for 3 min; (b) a 30-cycle PCR step, each cycle consisting of 95° C. for 30 s, 60° C. for 30 s, and 72° C. for 1 min; and (c) a final elongation step at 72° C. for 10 min. The barcode primers are listed in Table 1.
The desired PCR amplification products were electrophoresed through an agarose gel to excise bands of the desired size, and DNA was purified using a gel purification kit (AccuPrep™, Bioneer, Korea). For the construction of ˜1,000 bp DNA sequence, 3-8 gel-purified gene fragments were pooled. For each pool, restriction enzyme digestion was carried out as follows: when EarI or EcoP15I was used, 2 μl EarI or EcoP15I, 5 μl NEB buffer, 0.5 μl 100×BSA, 10 μl water, and 30 μl purified (and pooled) DNA fragments were mixed, followed by digestion at 37° C. for 3 h (for EcoP15I, 10 ATP was further added); and when BtsI was used, 2 μl BtsI, 5 μl NEB buffer, 0.5 μl 100×BSA, 10 μl water, and 30 μl PCR products were mixed, followed by digestion at 55° C. for 3 h. The restriction digest products were electrophoresed through 1.5% agarose gels to obtain expected bands (daughter fragments, 300 bp;
Nested PCR for 1 Kb DNA Synthesis Using Flanking Sequence Removed Shotgun Assembly Products
The flanking sequence removed shotgun assembly products were assembled to make 11 gene cluster fragments (645-1,325 bp). The target DNA sequences are listed in Table 4.
The 11 gene cluster fragments were constructed using 3 μl water, 10 μl Phusion polymerase pre-mix (NEB, MA), 1 μl forward and reverse primers, and 5 μl of flanking sequence-cleaved shotgun assembly DNA fragments (
Nested PCR Assembly of an 11.4 Kb Gene Cluster Using Flanking Sequence Removed Shotgun Assembly Products
A nested PCR method was used to assemble eleven ˜1 kb fragments into the full-length target penicillin biosynthetic gene cluster.
The PCR was performed using eleven ˜1 kb fragments (each 1 μl) and 15 μl of Phusion polymerase pre-mix (NEB, MA) in the absence of primers as follows: (a) a pre-denaturation step at 95° C. for 3 min; (b) a 10-cycle PCR step, each cycle consisting of 95° C. for 30 s, 70° C. for 30 s, and 72° C. for 3 min 30 s; and (c) a final elongation step at 72° C. for 5 min.
1 μl primer pairs containing restriction enzyme sites (BglII or NotI) were added to the mixture (˜1 kb fragments (each 1 μl) and 15 μl of Phusion polymerase pre-mix) and 25 more PCR cycles were performed. The PCR products were used for cloning.
After gel-electrophoresis, bands of the desired size were excised and DNA was purified. The products were cloned into a pBK3 vector (Kim, H., et al., 2010) using BglII and NotI restriction enzymes, and C2566 E. coli competent cells were transformed with the vector. After overnight growth at 37° C., a few colonies were screened for pBK3 vector containing the desired DNA insert size using colony PCR. Several colonies were grown in LB media for plasmid extraction using an AccuPrep™ plasmid extraction kit (Bioneer, Korea). The extracted plasmid was submitted for sequencing. Sequencing data were analyzed using the Lasergene program (DNAstar, Madison, Wis., USA).
Results and Discussion
The shotgun DNA synthesis technology was developed to overcome the challenges of high-throughput DNA construction. 228 oligonucleotides were designed to construct a penicillin biosynthetic gene cluster [N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase, 11,376 bp]. Chip oligonucleotides were designed to contain generic flanking sequences and cleaved from a 55K Agilent DNA microchip. Selective amplification was carried out using flanking sequences and amplification primer sequences were removed using the Type IIS restriction enzymes to obtain a sub-pool of chip oligonucleotides (
The key point for the success of the method of the present disclosure is based on the hypothesis that a pool of oligonucleotides can be shotgun assembled in one pot to produce heterogeneous assembly products, and that each one of these products can be identified by high-throughput sequencing. Thus, oligonucleotides, at least one end of which had been cleaved, were used for shotgun DNA synthesis. As expected, highly heterogeneous DNA fragments ranging in size from 100 bp to 1,000 bp were produced (
The present inventors then focused on developing a method to identify random fragment compositions using high-throughput sequencing technology, as well as a method to obtain sequence-validated error-free fragments from the pool of DNA fragments (
The tagging barcode primer sequences consisted of three parts containing the original primer sequences used for the amplification of DNA chip: (a) generic primer sequences used in designing oligonucleotides, (b) 20 bp degenerate-barcode sequences, and (c) 454 primer sequences. The barcode sequence-attached shotgun assembly fragments were further amplified using the 454 primer sequences to increase the concentration of the barcoded assembly products.
It was found that through 454 sequencing analysis of the shotgun assembly fragments, 3% of the DNA fragments (˜400 bp) were error-free (
This analysis using the Python program resulted in error-free shotgun assembled DNA fragments (˜300 bp) covering 88% of the 11,376 bp target sequence. For the remaining ˜12% DNA sequences, the error containing sequences were analyzed to determine which sequences could be re-amplified using primers. 61 pairs of PCR barcode primers were selected from a pool of random assembly products.
The desired shotgun assembly fragments were selectively amplified from the DNA mixtures using degenerate-barcode primer sequences. Based on the gel data (˜400 bp), 77% (47 out of 61) of selective amplification reactions resulted in the desired sequences. The non-amplified target sequences were re-evaluated through the Python program. As a result, alternative oligonucleotide sequences were ordered. The alternative primer sequences could be utilized to obtain 100% sequences, which could be used for target DNA synthesis. The sequences (˜10%) were TOPO cloned for Sanger DNA sequencing to evaluate their effectiveness. About 99.98% of the Sanger sequencing-evaluated sequences matched with the target reference sequence.
Amplicons using selected DNA include flanking sequences containing Type IIS restriction enzyme recognition sequences used in the processing procedure of chip oligonucleotides. Accordingly, prior to assembly of the amplified error-free fragments into the target DNA, the barcode sequences of the amplified fragments were cleaved with Type II restriction enzymes (Type IIS restriction enzyme, EarI, BtsI or EcoP 15I) (
It is worth to further discuss various points in order to illustrate the creative features of the present disclosure. First, the shotgun synthesis of the present disclosure can provide a solution to the intrinsic challenges associated with low DNA assembly efficiency. DNA assembly processes occur less efficiently due to the increased number of oligonucleotides in a sub-pool (causing a low oligonucleotide concentration) and the presence of partially cleaved flanking sequences in the oligonucleotides. For example, highly heterogeneous by-products of ˜100-500 bp corresponding to small-sized DNA fragments were observed continuously during assembly of target gene clusters. In contrast, the shotgun DNA synthesis of the present disclosure enables the use of highly heterogeneous by-products in subsequent DNA assembly processes and therefore has advantages over conventional gene synthesis methods.
Second, a method of identifying and isolating error-free DNA fragments from a number of random shotgun assembly products was successfully developed. Barcoded primer sequences of the synthetic DNA sequence were validated by high-throughput sequencing. The barcode sequences could be utilized in selective PCR amplification of desired DNA molecules from a pool of the DNA molecules. After removal of the amplification primer sequences from the selectively amplified target DNA fragments, the fragments were hierarchically used in the assembly of the target sequence. In addition, it is evident that when the size of the target DNA molecules is sufficient to be sequenced at one time by the next-generation sequencing technology, the products obtained in the first round of the shotgun synthesis can be directly used.
Third, a cost estimate for DNA synthesis using Agilent chip-oligonucleotides and high-throughput sequencing is provided below. The two major costs associated with synthesis of large DNA are the costs of oligonucleotides and sequencing. The synthesis cost of chip oligonucleotides is expected to be $0.00085/nt, which is 100 times cheaper than resin-based oligonucleotides (Kim et al., 2011). In addition, 454 sequencing reads were computationally analyzed for sequencing cost-analysis. As a result, it was confirmed that 3% of the 300 bp DNA fragments produced in the first round of shotgun synthesis were error-free. The sequencing reading was performed using ⅛ lane of Roche-454 sequencing, which costs about $1,500. That is, the cost of synthesizing the 10 kb gene cluster was close to $3,000 (the cost of synthesizing oligonucleotides=$ 0.00085/nt*2*228*150 nt=$60; and the cost of various primers=$0.1/nt*200*20 nt=$400; the cost of Sanger sequencing=$3*100 reaction=$300; Roche-454 sequencing cost=$1,500; the cost of various purification kits and enzymes=$800). The cost of DNA synthesis by the synthesis method of the present disclosure is at least five times lower than the current price ($0.5/bp) charged by DNA synthesis companies. The concern that the present inventors have with this approach is the uneven coverage of the DNA assembly fragments. From the repeated assembly experiments, the present inventors found that the coverage of certain regions from the DNA assembly processes was dependent on the DNA sequences. It would be ideal to develop a shotgun assembly process that provides more uniform coverage.
Although the particulars of the present disclosure has been described in detail, it will be obvious to those skilled in the art that such particulars are merely preferred embodiments and are not intended to limit the scope of the present disclosure. Therefore, the true scope of the present disclosure is defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2011-0076408 | Aug 2011 | KR | national |
This application is a National Stage Application of PCT International Patent Application No. PCT/KR2012/006147 filed on Aug. 1, 2012, under 35 U.S.C. §371, which claims priority to Korean Patent Application No. 10-2011-0076408 filed on Aug. 1, 2011, which are all hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2012/006147 | 8/1/2012 | WO | 00 | 1/29/2014 |
