KR101264295B1 - Hierarchial Gene Synthesis Methods of a Target Nucleic Acid Sequence - Google Patents

Abstract

The present invention provides a hierarchical method of preparing a target nucleic acid sequence of 1 kb or more. The method of the present invention effectively utilizes oligonucleotides synthesized from DNA microchips comprising flanking DNA sequences composed of multiple layers to effectively synthesize target nucleic acid sequences of 1 kb or more, which were not possible with conventional methods. It can be carried out. In addition, the cost of gene synthesis through the method of the present invention is more than three times cheaper than conventional methods using resin-based oligonucleotides. Thus, the method of the present invention provides a very good means to significantly reduce the cost of gene synthesis.

Description

Hierarchial Gene Synthesis Methods of a Target Nucleic Acid Sequence

The present invention relates to a method for hierarchically preparing a target nucleic acid sequence of 1 kb or more.

Techniques for constructing large DNA stretches (ie, 200 bp to full genome-sized DNA) can be applied to many areas of chemistry and biology (Czar et al. al . , 2009; Mueller et al . , 2009). For example, many protein synthesis methods to probe the biological system has been developed for the encoding sequence, and build the virus and having novo (de novo) of the microbial genome (Cello et al . , 2002; Gibson et al . , 2008; Gibson et al . 2010; Hutchison et al . , 2005; Kodumal et al . , 2004; Tian et al . , 2009; Tumpey et al . , 2005). Recently, synthesis of attenuated viral genomes for vaccine development (Coleman et al. al . , 2008; Mueller et al . , 2010), and the synthesis of codon-optimized antibody sequences for effective biological drug production (Welch et al . , 2009) further demonstrates the usefulness of DNA construction for medical applications.

Long DNA sequences can be assembled via polymerase chain reaction (PCR) using multiple overlapping synthetic oligonucleotides and excessive amounts of flanking primer sequences (Stemmer et. al . , 1995). In addition, effective assembly can be performed by a ligase chain reaction (LCR) combined with PCR (Smith et al. , 2003). However, the high-cost and high synthetic error rates of oligonucleotides (Bang and Church, 2008; Smith et. al . , 2003) are two major barriers to low-cost gene synthesis. For this reason, the development of low-cost techniques for DNA synthesis is urgently required.

Currently, there are two different approaches for low cost oligonucleotide synthesis for the synthesis of long DNA sequences. In a first approach, the Quake group assembled 200 bp DNA molecules using conventionally controlled pore glass resin and microfluidic devices to synthesize 16 oligonucleotides (Lee et al . , 2010). This approach has the advantage that the synthetic oligonucleotides from the microfluidic device can be used for assembly without further amplification. However, the scalability of the approach is still limited because only a small number of oligonucleotides can be synthesized from the device.

In another effort to develop low-cost DNA synthesis methods, Tian et al. Designed a method using DNA oligonucleotides derived from DNA microchips that can be cleaved (Tian et al., 2004). Hundreds of oligonucleotides were synthesized, cleaved and amplified on programmable Atactic chips (Houston, TX), after which the oligonucleotides were assembled into 21 genes encoding E. coli ribosomal proteins (Tian et al. , 2004). To construct multiple DNA molecules, similar synthetic methods were carried out using DNA microchips (Richmond et. al . , 2004; Zhou et al . , 2004). More recently, block-based gene assembly methods using microarray oligonucleotides have been proposed (Borovkov et al. , 2010). Surprisingly, the block-based synthesis strategy simplifies the gene synthesis process by eliminating the need for an oligonucleotide amplification step. However, all the microarray-based gene synthesis methods described above have been limited to the construction of small sized DNA molecules (less than 1 kb), which makes it difficult to construct long DNA molecules using microchip-derived oligonucleotides. This is because: the construction of 14.6 kb DNA assembled using 21 DNA fragments was a very exceptional case (Tian et. al . , 2004).

Therefore, there is an urgent need in the art for a method for effectively assembling longer DNA molecules.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a novel method of synthesizing a target nucleic acid sequence of 1 kb or more more economically and efficiently. As a result, the inventors have used oligonucleotide pools derived from DNA microchips comprising flanking DNA sequences consisting of multiple layers containing multiple common primer sequences and at least two restriction enzyme recognition sequences. The present invention has been completed by discovering that a target nucleic acid sequence of kb or more can be synthesized.

It is an object of the present invention to provide a method for producing a hierarchical preparation of a target nucleic acid sequence.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for making a hierarchical preparation of a target nucleic acid sequence comprising the following steps: (a) 5'-end first common priming in the 5 'to 3' direction Comprising a generic priming sequence, a first 5'-restriction cleavage sequence, an overlapping target portion sequence, a first 3'-restriction cleavage sequence, and a 3'-terminal first common priming sequence Preparing an oligonucleotide pool; Some of the overlapping target sequences in the oligonucleotide pool comprise the entire target nucleic acid sequence when they overlap each other, and the primary 5′-limiting enzyme cleavage sequence and the primary 3′-limiting enzyme cleavage sequence of the oligonucleotide Are the same as each other; The first oligonucleotide for generating the 5'-terminal portion of the first intermediate product in the oligonucleotide pool is a 5'-second common priming sequence in the 5 'to 3' direction downstream of the 5'-restriction cleavage sequence. And a second oligonucleotide to further generate a 3'-terminal portion of the first intermediate product, wherein the second oligonucleotide further comprises a 3 'to 5' upstream of the 3'-restriction cleavage sequence. Additionally a 3'-second common priming sequence and a second 3'-restriction cleavage sequence in the direction; The third oligonucleotide for generating the 5-terminal portion of the second intermediate product is a 5'-third common priming sequence and a third 5 'in the 5' to 3 'direction downstream of the 5'-restriction cleavage sequence. A fourth oligonucleotide comprising a restriction enzyme cleavage sequence, wherein the fourth oligonucleotide for generating the 3'-terminal portion of the second intermediate is 3'-third in a 3 'to 5' direction upstream of the 3'-restriction cleavage sequence Further comprises a common priming sequence and a tertiary 3′-limiting enzyme cleavage sequence; (b) amplifying the oligonucleotide pool with a polymerase chain reaction (PCR) using a first primer set that hybridizes to the 5'-terminal first common priming sequence and the 3'-terminal first common priming sequence; (c) cleaving the amplification product of step (b) with a restriction enzyme that acts on the first 5′-limiting enzyme cleavage sequence and the first 3′-limiting enzyme cleavage sequence; The cleavage causes the 5′-terminal first common priming sequence and 3′-terminal first common priming sequence to be released from the pool into the oligonucleotides; A second common priming sequence is located at the 5'-end of the first oligonucleotide, a second common priming sequence is located at the 3'-end of the second oligonucleotide, and a 5'-end of the third oligonucleotide. A third common priming sequence is located, and a third common priming sequence is located at the 3′-end of the fourth oligonucleotide; (d) a first assembly amplification reactant 1 comprising (i) a set of primers hybridizing to a second common priming sequence of the first oligonucleotide and a second common priming sequence of the second oligonucleotide and (ii) the third A first assembly amplification reaction was carried out using a first assembly amplification reactant 2 comprising a primer set hybridized to a third common priming sequence of the oligonucleotide and a third common priming sequence of the fourth oligonucleotide to obtain a first intermediate product. intermediates) and a second intermediate product; (e) replacing the first intermediate product and the second intermediate product of step (d) with (i) the second 5′-limiting enzyme cleavage sequence of the first oligonucleotide and the second 3 ′ of the second oligonucleotide. A restriction enzyme that acts on the restriction enzyme cleavage sequence and (ii) a third 5'-limiter cleavage sequence of the third oligonucleotide and a third 3'-limiter cleavage sequence of the fourth oligonucleotide Cleaving with restriction enzymes; And (f) assembling the result of step (e) to obtain a target nucleic acid sequence.

The present inventors have tried to develop a novel method of synthesizing a target nucleic acid sequence of 1 kb or more more economically and efficiently. As a result, the present inventors have found that a target nucleic acid sequence of 1 kb or more can be synthesized using an oligonucleotide pool derived from a DNA microchip comprising flanking DNA sequences composed of multiple layers.

The present invention provides a method for synthesizing a target nucleic acid sequence using a DNA microchip-derived oligonucleotide, which is more economically and efficiently synthesized than 1 kb or more target nucleic acid sequences. In the method of the present invention, oligonucleotides of the present invention can be used to scale up larger genes (> 1 kb).

Preferably, the oligonucleotide pools used in the present invention are derived from DNA microchips, wherein the oligonucleotides comprise multiple layers of DNA flanking sequences.

According to a preferred embodiment of the invention, the oligonucleotides of the invention are 5'-terminal first common priming sequence, primary 5'-restriction cleavage sequence, overlapping target partial sequence, primary 3 ' A restriction enzyme cleavage sequence and a 3'-terminal first common priming sequence.

The first common primer sequence pairs present at both ends of the oligonucleotides are for amplifying the amount of oligonucleotides derived from a DNA chip, and primers for producing a sufficient amount of double-stranded-oligonucleotides. Used as an annealing position of the set.

As used herein, the term nucleotide is a deoxyribonucleotide or ribonucleotide that exists in single- or double-stranded form and includes analogs of natural nucleotides unless otherwise specified (Scheit, Nucleotide). Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

According to a preferred embodiment of the invention, the gene amplification of the invention is carried out by PCR. According to a preferred embodiment of the present invention, the primers of the present invention are used for gene amplification reactions.

The term primer, as used herein, refers to oligonucleotides, conditions under which the synthesis of primer extension products complementary to the nucleic acid chain (template) is induced, i.e., the presence of polymerizers such as nucleotides and DNA polymerases, and suitable temperatures. It can serve as the starting point of synthesis under the condition of and pH. Preferably, the primer is a deoxyribonucleotide and is a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.

The primer should be long enough to prime the synthesis of the extension product in the presence of a polymerizer (eg DNA polymerase). Suitable lengths of primers are typically 15-30 nucleotides, although varying depending on a number of factors, such as temperature, application and source of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template. According to a preferred embodiment of the present invention, the primer of the present invention is produced using the Per1 program which is a computer program.

As used herein, the term annealing or priming refers to the placement of oligodioxynucleotides or nucleic acids into a template nucleic acid, wherein the polymerase polymerizes the nucleotides to form a nucleic acid molecule that is complementary to the template nucleic acid or portion thereof. To form. The term hybridization, as used herein, means that two single stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization refers to the complementarity between single stranded nucleic acid sequences. This perfect match may occur or even if some mismatch base is present The degree of complementarity required for hybridization may vary depending on the hybridization reaction conditions, and in particular may be controlled by temperature. As used herein, the term annealing and hybridization do not differ and are used interchangeably herein.

According to the method of the present invention, the present invention performs restriction enzyme cleavage after amplification of the oligonucleotide pool using a primer set hybridized to the first common priming sequence. Subsequently, assembly PCR for producing a first intermediate product and a second intermediate product in the oligonucleotide pool is performed by reacting with each other independently from each other. Finally, assembly PCR of the first intermediate product and the second intermediate product is performed to prepare a target nucleic acid sequence of 1 kb or more.

According to a preferred embodiment of the present invention, the overlapping target portion sequence includes the entire target nucleic acid sequence when overlapping each other.

The present invention uses intermediates to synthesize target nucleic acid sequences.

According to a preferred embodiment of the invention, the intermediates are synthesized independently of each other.

More specifically, the first intermediate product of the present invention is prepared by assembly PCR of oligonucleotides amplified in step (a). The oligonucleotides comprise sequences that overlap each other. The overlapping sequence refers to the complementary sequence of some of the oligonucleotide sequences, which is designed to enable sequential assembly of oligonucleotides to produce the first intermediate product. Preferably, the first oligonucleotide for generating the 5'-terminal portion of the first intermediate product comprises a 5'-second common priming sequence downstream of the 5'-restriction cleavage sequence in a 5 'to 3' direction. The second oligonucleotide, further comprising a second 5'-restriction cleavage sequence, for generating the 3'-terminal portion of the first intermediate product, is directed 3 'to 5' upstream of the 3'-restriction cleavage sequence. Further comprising a 3'-second common priming sequence and a second 3'-restriction cleavage sequence. In addition, several oligonucleotides constituting the first intermediate product present between the first oligonucleotide and the second oligonucleotide contain only overlapping sequences that are sequentially assembled, Flanking sequences are not included. In addition, the third oligonucleotide for generating the 5′-terminal portion of the second intermediate may also be formed by the 5′-third common priming sequence and the 3 ′ in the 5 ′ to 3 ′ direction downstream of the 5′-restriction cleavage sequence. The fourth oligonucleotide, which further comprises a primary 5'-restriction cleavage sequence, to generate the 3'-terminal portion of the second intermediate product, is a 3 'to 5' direction upstream of the 3'-restriction cleavage sequence. Additionally comprises a '-third common priming sequence and a third tertiary' restriction enzyme cleavage sequence.

According to a preferred embodiment of the present invention, both ends of the first intermediate product of step (a) are composed of the first oligonucleotide and the second oligonucleotide in the 5 'to 3' direction, the second step of (a) Both ends of the intermediate product consist of the third oligonucleotide and the fourth oligonucleotide in the 5 'to 3' direction.

According to a preferred embodiment of the present invention, the number of oligonucleotides forming the first intermediate product and the second intermediate product of step (a) may be determined according to the length of the target nucleic acid sequence. For example, the number of oligonucleotides forming the first intermediate product and the second intermediate product used in the synthesis of the target nucleic acid sequence of 1 kb or more is at least 3, more preferably at least 4, and even more Preferably at least five.

According to a preferred embodiment of the present invention, the second restriction enzyme cleavage sequence and the third restriction enzyme cleavage sequence of step (a) are the same or different, more preferably different.

According to a preferred embodiment of the invention, the amplification of the first intermediate product and the second intermediate product of step (b) is carried out in different tubes.

According to a preferred embodiment of the present invention, the size of the first intermediate product and the second intermediate product of step (b) is 300-500 bp, more preferably 400-500 bp.

The term complementary, as used herein, means having complementarity sufficient to selectively hybridize to the above-described nucleotide sequence under certain specific hybridization or annealing conditions, and substantially complementary. And perfectly complementary, and preferably completely complementary.

The term amplification reaction described herein means a reaction that amplifies a target nucleic acid molecule. A variety of amplification reactions have been reported in the art, including PCR (PCR) (US 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. The method of Miller, HI (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 19, WO 88 / 10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence (CP-PCR) (US Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (US Pat. No. 5,413,909 and 5,861, 245), nucleic acid sequence based amplification ions; NASBA (US Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603) and strand displacement amplifications 21, 22. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

In the most preferred embodiment of the present invention, the amplification process is carried out according to the PCR disclosed in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.

PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

The target nucleic acid sequence that can be used in the present invention is not particularly limited, and preferably includes DNA (gDNA or cDNA) and RNA, and more preferably DNA. In addition, target nucleic acid molecules include, for example, prokaryotic nucleic acids, eukaryotic cells (eg, protozoa and parasites, fungi, yeast, higher plants, lower animals, and higher animals, including mammals and humans) nucleic acids, viruses (eg, herpes) Virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, poliovirus, etc.) nucleic acid or non-loid nucleic acid.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

Various DNA polymerases can be used for amplification of the present invention and include Klenow fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which is 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 DNA polymerase of Thermosipho africanus .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with such joins as Mg ^{2 +} , dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables. This amplified oligonucleotide pool can be used to assemble a target nucleic acid sequence of 1 kb or more.

According to a preferred embodiment of the invention, the assembly PCR carried out in the method of the invention is carried out at a high annealing temperature, more preferably at 65 ℃ or more, most preferably at 65-72 ℃.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a hierarchical production method for more economically and efficiently synthesizing a target nucleic acid sequence of 1 kb or more.

(b) The method of the present invention utilizes oligonucleotides comprising flanking DNA sequences consisting of multiple layers.

(c) The method of the present invention enables the synthesis of target nucleic acid sequences of 1 kb or more, which were not possible with the conventional methods.

(d) In addition, the cost of gene synthesis through the method of the present invention is more than three times lower than conventional methods using resin-based oligonucleotides.

(e) Thus, the method of the present invention provides a very good means by which the cost of gene synthesis can be significantly reduced.

1 is a schematic diagram illustrating a method of synthesizing the Dpo4 gene by two different synthesis strategies. (A) shows the process of assembling three fragments into one gene. (B) The process represents direct assembly into the target gene.
2 is a gel photograph of the process of building the Dpo4 gene. The band of the expected product is indicated by a red triangle. 2A is a 6% TBE gel photograph of Agilent chip oligonucleotides PCR-amplified with flanking primers. Lane 1, 100 bp DNA ladder; Lane 2, 10 bp DNA ladder; And lane 3, Agilent chip oligonucleotide. Figure 2b is a photo of the amplified ds-oligonucleotide digested with Ear I restriction enzyme and electrophoresed on 6% TBE gel. Figure 2c is the result of the first assembly PCR to form three fragments for the Dpo4 gene. Lane 1, 100 bp DNA ladder; Lane 2, first fragment of 363 bp; Lane 3, second fragment of 429 bp; And lane 3, third fragment of 330 bp. FIG. 2D is an agarose gel photograph of the Dpo4 gene assembled using the three DNA fragments shown in FIG. 2C. 2E shows oligonucleotides for direct synthesis of Dpo4 gene amplified using flanking primers. Figure 2f is the result of amplified ds-oligonucleotides were digested with Ear I restriction enzyme, and then loaded into lane 2 and electrophoresed. 2G is a gel photograph showing the one-pot synthesis of the Dop4 gene.
3 is a gel image result showing the hierarchical synthesis of the Dpo4 gene. 3A is a 6% TBE gel photograph of ds-oligonucleotides amplified with flanking primer pairs. Lane 1, 10 bp DNA ladder; And lane 2, PCR product. Figure 3b is the result of electrophoresis after amplified ds-oligonucleotides were cleaved with Ear I restriction enzyme. Lane 1, 100 bp DNA ladder; And lane 2, cleavage product. Figure 3c is the result of loading the 600 bp intermediate fragments for the Dpo4 gene in the agarose gel. Lane 1, 100 bp DNA ladder; And lane 2, intermediate. 3D shows the final assembled PCR product, with the Dpo4 product loaded in the second lane.
4 is a diagram schematically illustrating a hierarchical gene synthesis method. Initial PCR amplification of Agilent chip oligonucleotides was performed in two different tubes. After removal of the generic adopter sequences, the ds-oligonucleotides were used to construct 400-500 bp DNA fragments. Two sets of replicable flanking sequences were used to construct various 400-600 bp DNA fragments simultaneously in one-pot. A second set of flanking sequences was cut from the intermediate fragments, and then a pool of intermediate fragments was used to construct the target gene.
5 is a gel photograph of the process of synthesizing the hierarchical Pfu gene. (a) Agilent chip oligonucleotides were amplified using two pairs of primers in two different PCR tubes. Lane 1, 10 bp DNA ladder; Ds- oligonucleotides having lane 2, Ear I and Bsg I restriction enzyme positions; And ds-oligonucleotides having lane 3, Ear I and Bts I restriction enzyme positions. (b) The flanking sequences of the two ds-oligonucleotides were cleaved with Ear I restriction enzymes. (c) Assembly PCR was performed using a second set of flanking primers. Lane 1, 100 bp DNA ladder; Lane 2, 4 fragments having Bsg I restriction enzyme positions; And lane 3, 3 fragments having Bts I restriction enzyme positions. (d) Two different sets of fragments were digested with Bsg I or Bts I, followed by final assembly PCR. The long Pfu gene of 2,325 bp was successfully produced. Lane 1, 2-log DNA ladder; And lane 2, assembled PCR product.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Experimental material

Unless specifically stated, all materials were purchased from the vendors listed below. DNA microarray oligonucleotides were purchased from Agilent (Santa Clara, CA, USA). Other oligonucleotides and primers were purchased from IDT (Coralville, IA, USA), Bioneer (Korea) and Macrogen (Korea). DNA purification kits and plasmid extraction kits were purchased from Qiagen (Valencia, CA, USA) and Bioneer (Korea). Restriction enzymes and ligase were purchased from NEB (New England Biolabs, Mass., USA), Fermentas and Takara (Japan). iProof ^™ DNA polymerase was purchased from Bio-rad (Hercules, CA, USA). DNA sequencing analysis program (Lasergene) was purchased from DNAstar (Madison, WI, USA).

Target DNA Sequencing and Oligonucleotide Design

Codon-optimized Dpo4 (1,056 bp; protein sequence from Sulfolobus solfataricus P2 DNA polymerase IV) and Pfu DNA polymerase (GenBank accession number, P61875) were selected as synthetic models. To design the positive and negative strand oligonucleotides, we used an in-house Per1 program. Each Agilent chip oligo comprising a generic flanking sequence was 150 nucleotides in length. The oligonucleotides were synthesized by Agilent in the form of microarrays. Oligonucleotide sequences are provided in the target sequence information below.

One-pot Dpo4 synthesis

A 150 bp double stranded DNA sequence was prepared by PCR amplification using flanking primers and Agilent chip oligonucleotides cleaved from microarrays.

The PCR was carried out with 50 reactants using iProof ^™ DNA polymerase (Bio-rad) according to the manufacturer's protocol, and the conditions were as follows: (a) a premodification step, 3 min at 95 ° C .; (b) 25 cycles of a PCR step consisting of 30 seconds at 95 ° C., 30 seconds at 57 ° C. and 1 minute at 72 ° C .; And (c) final extension step, 10 minutes at 72 ° C. The PCR product was then stored at 4 ° C. We re-amplified the PCR product to increase oligonucleotide concentration. The PCR mixture contained 200 μl iProof ^™ DNA polymerase, 160 μl water, 8 μl first PCR product, 10 μl forward primer (20 M) and 10 μl reverse primer (20 μM). PCR conditions were the same as those of the first PCR except for 10 cycles of PCR amplification. We purified the product using a PCR purification kit and eluted with 45 μl of water (desalting and buffer exchange). The purified ds-oligonucleotides were cleaved with Ear I restriction enzymes for 37 to 3 hours. The reaction mixture was prepared by mixing 45 μl of kit-purified product, 10 μl of Ear I (20 units / μl; NEB) restriction enzyme, 0.5 μl of BSA and 6 μl of 10 NEB buffer 1. Next, we electrophoresed 6% TBE gels to determine whether the ds-oligonucleotides were cleaved correctly. The inventors then purified the product using a PCR purification kit. The ds-oligonucleotide pool was subjected to assembly PCR using a primer having Xba I and Pst I restriction enzyme positions as a template. The total reaction capacity was 20 μl and was identical to the PCR conditions described above except for annealing temperature of 72 ° C. and 35 cycles of PCR amplification. We purified the final assembly product via agarose-gel purification. The purified product was cloned into pUC19 vector using Xba I (20 units / μl; NEB) and Pst I (20 units / μl; NEB) restriction enzymes. After performing restriction enzyme digestion for 2 hours, the inventors performed DNA column purification to stop enzyme activity and eluted the product with 20 water. We added 2 μl of T4 DNA ligation buffer and 2 μl of T4 DNA ligase (400 units / μl; NEB) to the mixture and reacted overnight at 16 ° C., followed by Competent E. Derived from NEB C2987. Coli cells were chemically transformed. After growing colonies overnight at 37 ° C. in agar plates containing carbenicillin antibiotics, we performed colony sequencing. Sequencing data was analyzed using a Lasergene.

Dpo4 synthesis through three fragment assemblies

Unlike the strategy used for One-pot Dpo4 synthesis, each Agilent chip oligonucleotide contained Ear I and Bcc I restriction enzyme positions in the flanking sequences. The inventors performed PCR amplification of oligonucleotides using flanking primers according to the same PCR conditions used for One-pot Dpo4 synthesis. Re-amplification procedures (10 cycles of PCR) were performed to increase the oligonucleotide concentration. We purified ds-oligonucleotide products using PCR purification kits. Restriction digestion was performed using Ear I and Bcc I restriction enzymes.

The reaction mixture was 45 μl of PCR product, 5 μl of Ear I (20 units / μl; NEB) restriction enzyme, 5 μl of Bcc I (20 units / μl; NEB) restriction enzyme, 6 μl of 10 NEB buffer 1 and 0.5 Prepare by mixing μl of BSA (20 ×). Next, we electrophoresed 6% TBE gels to identify 100 bp of well-cut ds-oligonucleotides. PCR purification kits were used to obtain processed ds-oligonucleotides. We ran the first assembly PCR in different PCR tubes with expected sizes of 363 bp, 429 bp and 330 bp for each reaction. We electrophoresed 3 μl of PCR product in 6% TBE gel in each tube to assess the size of the product and cut the band to break the gel. The crushed gel was dissolved in 50 μl of TE buffer and reacted at 65 ° C. for DNA extraction. To assemble the Dpo4 gene using primers with Xba I and Pst I restriction enzyme positions, we performed assembly PCR of these three fragments. The present inventors electrophoresed the PCR product in an agarose gel to cut a 1 kb size region to perform gel purification using a gel extraction kit and clone the product into a pUC19 vector using Xba I and Pst I restriction enzymes. It was. We performed DNA column purification after restriction enzyme digestion for 2 hours and added T4 DNA ligation buffer and T4 DNA ligase. The mixture was reacted overnight at 16 ° C., and then chemically transformed into competent E. coli cells. After growing colonies overnight at 37 ° C. in agar plates, we selected colonies for colony sequencing.

Hierarchical ( Hierarchical ) Dpo4  synthesis

PCR amplification was carried out using oligonucleotides cleaved from Agilent chips containing the first set of flanking primer sequences as templates to produce 150 bp of product. PCR reaction conditions were as follows: (a) a total denaturation step, 95 min at 95 ° C .; (b) 26 cycles of PCR consisting of 30 seconds at 95 ° C., 30 seconds at 53 ° C. and 1 minute at 72 ° C .; And (c) final extension step, 10 minutes at 72 ° C. The PCR product was then stored at 4 ° C. and the PCR product was re-amplified (10 cycles of PCR amplification) under the same PCR conditions as above except for the annealing temperature of 53 ° C. to increase the oligonucleotide concentration. Next, the inventors were electrophoresed on 6% TBE gel and mixed with 45 μl of PCR product, 10 μl of Ear I restriction enzyme, 6 μl of 10 NEB buffer and 0.5 μl of BSA to react at 37 ° C. Restriction enzyme cleavage was performed. Thereafter, assembly PCR was performed using a second set of flanking primers. The conditions of the assembly PCR were as follows: (a) a total denaturation step, 95 min at 95 ° C .; (b) 35 cycles of a PCR step consisting of 30 seconds at 95 ° C., 30 seconds at 72 ° C. and 1 minute at 72 ° C .; And (c) final extension step, 10 minutes at 72 ° C. The PCR product was stored at 4 ° C. The restriction enzyme cleavage was then performed using EarI restriction enzymes to cleave the flanking sequences (even though we could use Ear I in both primer sequences of the first and second PCRs) The use of the type II restriction enzyme sites of may improve the synthetic efficiency: see, The following method for hierarchical synthesis of genes encoding Pfu DNA polymerase.

Assembly PCR was performed using primers with Xba I and Pst I restriction enzymes, linking oligonucleotides and iProof ^™ DNA polymerase. The PCR reaction mixture contained 10 μl of iProof ^™ DNA polymerase, 9 μl of water, 1 μl of restriction enzyme cleaved product, 0.5 μl of forward primer, 0.5 μl of reverse primer and 1 μl of linking oligonucleotide (0.4 μM). Included. PCR was carried out with 25 cycles of PCR amplification at annealing temperature of 63 ° C.

We electrophores the PCR products on agarose gels to perform gel purification using a gel extraction kit and clone the products into pUC19 vectors using Xba I and Pst I restriction enzymes. Cloning and transformation steps were performed as described above. Then, we selected colonies and performed colony sequencing.

Hierarchical Pfu  synthesis

PCR amplification was performed using a template of oligonucleotides cleaved from Agilent chips with two sets of flanking primers in two different tubes producing 150 bp of product. PCR reaction conditions were as follows: (a) a total denaturation step, 95 min at 95 ° C .; (b) 26 cycles of PCR consisting of 30 seconds at 95 ° C., 30 seconds at 53 ° C. and 1 minute at 72 ° C .; And (c) final extension step, 10 minutes at 72 ° C. The PCR product was stored at 4 ° C. The PCR product was then re-amplified in the same manner as the PCR conditions described above except for the annealing temperature of 53 ° C. to increase the oligonucleotide concentration. Electrophoresis was performed on 6% TBE gels to identify the product, and 90 μl of PCR product, 10 μl of Ear I restriction enzyme, 10 μl of 10 NEB buffer 1 and BSA were mixed and reacted at 37 ° C. for 3 hours. Enzyme cleavage was performed to remove flanking sequences. After DNA column purification (eluted with 40 μl of water), fragment PCR of the pfu DNA polymerase gene was made in two different reaction mixtures by assembly PCR using a second set of flanking primers. Assembly PCR conditions were the same as the PCR conditions described above except that a 35 cycle PCR amplification procedure was performed at an annealing temperature of 60 ° C. The PCR reaction mixture contained 10 μl of iProof ^™ DNA polymerase, 4 μl of water, 6 μl of flanking sequence-cleaved ds-oligonucleotides, 0.5 μl of forward primer and 0.5 μl of reverse primer. PCR was carried out with 25 cycles of PCR amplification at annealing temperature of 63 ° C. Restriction digestion was performed at 37 ° C. for 1 hour in one tube containing 2 μl of Bsg I restriction enzyme (5 units / μl; NEB), 14 μl of PCR product, 2 μl of NEB buffer 4 and 1 × SAM. . In addition, restriction enzyme cleavage in the second tube was performed for 2 hours at 55 ° C. by mixing 2 μl of Bts I restriction enzyme (10 units / μl; NEB), 14 μl of PCR product, 2 μl of NEB buffer 1 and 1 × BSA. Was carried out. After purification of the DNA column, two reaction mixtures were each mixed with 6 µl, and the final assembly PCR was performed using primers having Xba I and Pst I restriction enzyme positions as templates. The PCR reaction mixture contained 10 μl of iProof ^™ DNA polymerase, 9 μl of water, 12 μl of purified pfu gene fragment, 0.25 μl of forward primer and 0.25 μl of reverse primer. PCR reaction conditions were as follows: (a) a total denaturation step, 95 min at 95 ° C .; (b) 25 cycles of PCR consisting of 30 seconds at 95 ° C., 30 seconds at 65 ° C. and 2.5 minutes at 72 ° C .; And (c) final extension step, 10 minutes at 72 ° C. The PCR product was stored at 4 ° C. We electrophores the PCR products on agarose gels to perform gel purification using a gel extraction kit and clone the products into pUC19 vectors using Xba I and Pst I restriction enzymes. Cloning and transformation steps were performed as described above. After growing colonies overnight at 37 ° C. in agar plates, we selected colonies for colony sequencing.

Experiment result

Oligonucleotides were cleaved from a 55K Agilent microarray (containing 55,000 independent oligonucleotide spots per chip). In this array, we produced 10,000 independent oligonucleotides (150 bp) in an amount of fmol using a proprietary ink-jet printing technique (LeProust et al.). Only a couple of hundreds of spots from the 55K chip were used in our experiments: the spots used 22 independent oligonucleotides in three pairs for hierarchical Dpo4 synthesis and one-pot Dpo4 synthesis. Twenty four independent oligonucleotides were used in three pairs, and 70 oligonucleotides were used in three pairs for the preparation of the pfu DNA polymerase gene.

Based on our previous work (Bang and Church, 2008), we evaluated the minimum concentration of each oligonucleotide for 1 kb gene synthesis in the range of 0.2 μM to 0.025 μM. Given that typical PCR was performed with 20 μl of reaction, we needed a minimum of 1 pmol (calculated as 0.05 M * 20 = 1 pmol) per oligonucleotide for 1 kb gene synthesis. However, the total oligonucleotide amount provided from the Agilent chip was 10 pmol (0.2 fmol per oligo). Thus, we diluted the oligonucleotide pool to 100 μl. As a result, the concentration of each oligonucleotide was 2 pM. That is, even when the entire Agilent chip oligonucleotide is used for one DNA synthesis reaction, the amount of each chip oligonucleotide (0.2 fmol) is estimated to be several thousand times less than the optimal amount of oligonucleotide (1 pmol) for one gene synthesis. . Since the concentration of chip oligonucleotides is too low to perform DNA assembly, prior to use in the Dpo4 and pfu DNA polymerase gene assemblies, we have identified replicable flanking sequences to selectively amplify oligonucleotide sub-pools of 55K chips. Was used (Tian et al . , 2004).

Obtaining sufficient amounts of replicable sequence-cleaved oligonucleotides is the most important factor for successful assembly of genes. To increase the concentration of flanking sequence-cleaved oligonucleotides, we assembled genes using double-stranded oligonucleotides (ds-oligonucleotides) instead of single-stranded oligonucleotides. The method of the present invention obviates the need for further processing from ds-oligonucleotides to ss-oligonucleotides, and elimination of this ss-oligonucleotide production step increases the total concentration of oligonucleotides. In addition, we have optimized a protocol that can directly use ds-oligonucleotides after type IIS restriction enzyme digestion without additional PAGE or agarose gel purification. By eliminating the gel purification step, we were able to obtain substantially large amounts of oligonucleotides.

Unlike typical gene synthesis methods of assembling 1 kb sequences into a one-step polymerase cycling assembly (PCA) using column-synthesized oligonucleotides, for preparing 1 kb genes using DNA chip oligonucleotides Initial attempts by the inventors confirmed that the assembly processes were insufficient (results not shown). Challenges in assembly with DNA sequences of 500 bp or more are associated with insufficient removal of the flanking regions of ds-oligonucleotides prior to assembly. In addition, non-uniform concentrations of ds-oligonucleotides derived from amplification of chip-cleaved oligonucleotides may contribute to inefficient gene synthesis. Thus, for the assembly of genes of 1 kb or more, we focused primarily on assembling the 1 kb Dpo4 gene using three fragments of about 350 bp. We used flanking sequence-cleaved oligonucleotides to assemble three Dpo4 fragments in three different reaction mixtures (FIGS. 1A and 2A-2C). The target fragments were designed to include overlapping regions. Thus, we used a pair of assembly primers to combine the three fragments during the second round of assembly to form the desired Dpo4 gene (FIGS. 1A and 2D).

In repeated experiments for the assembly of Dpo4 fragments, the inventors found that high annealing temperatures 65-72 during the DNA assembly increased synthesis efficiency (FIG. 3). The present inventors hypothesized that high annealing temperatures could contribute to an increase in assembly efficiency by excluding insufficiently cleaved oligonucleotides in the assembly process. Therefore, the inventors conducted assembly experiments at various assembly temperatures to investigate the possibility of producing DNA molecules of 1 kb or more (results not shown). As expected, we were able to assemble the Dpo4 gene at increased annealing temperatures (Figures 1B and 2E-2G). The inventors then cloned and sequenced the synthesized constructs (Table 1, sequencing data of one-pot synthesized Dpo4).

Comparison of sequence errors generated during the synthesis of target genes. Gene name
(Gene length)
Way
error ^a
Sequenced
gun bp
Error rate
(Error / 1 kb )
Dpo4 DNA Polymerase
(1,056 bp)
Dpo4 DNA Polymerase
(1,056 bp)
Pfu DNA Polymerase
(2,325 bp)
One-pot synthesis

Hierarchical synthesis

Hierarchical synthesis
15 (11: 1: 1: 2)

13 (8: 4: 1: 0)

22 (15: 2: 4: 1)
8,448

2,112

4,650
Approximately 1.8

6.2

About 4.7

^a error [deletion: insert: transition: transversion]

Although we have successfully synthesized the Dpo4 gene by the one-pot method using DNA chip-derived oligonucleotides, we have found that the synthesis efficiency is still low (see gel data shown in Figure 2g). It may also be argued that the increased length of the synthetic Dpo4 gene is only the increased length of the oligonucleotides provided by the Agilent chip. Accordingly, the inventors have sought to develop a scalable and hierarchical synthesis method that can be applied to the preparation of large DNA molecules. Our synthesis scheme is outlined in FIG. 4. The uniqueness of the synthetic method of the present invention is the design and introduction of multiple layers of flanking DNA sequences for the assembly of large DNA constructs. The primer sequence of the first layer amplifies the microchip-cut ss-oligonucleotides to produce ds-oligonucleotides. The ds-oligonucleotides were used for the preparation of 400-500 bp DNA fragments that could function as intermediates for target gene synthesis. According to the synthetic design, we additionally introduced the second layer of the replicative sequence into the intermediate 400-500 bp fragment sequences. The primers of the second layer allowed simultaneous production of multiple 400-500 bp DNA fragments in a one-pot method. The replicable flanking sequences contain type II restriction enzyme sites that are cleaved before the second round of assembly. As a result, we can use intermediate fragments to prepare target genes.

For successful hierarchical synthesis, we had to achieve assembly of multiple synthetic target DNA molecules in a one-pot method. To test whether multiple DNA fragments (approximately 500 bp) can be produced using single universal primer pairs in the same reaction mixture, we have two fragments without regions overlapping the Dpo4 gene. (528 bp each). Using common primer pairs, we prepared both fragments in the same reaction mixture (FIG. 3). Identification of the synthetic fragments via DNA sequencing confirmed that our method was reliable (results not shown). Furthermore, the inventors were able to link the two fragments simply by using a linking oligonucleotide and performed sequencing based on this (sequencing data of Dpo4 synthesized hierarchically in FIG. 3 and Table 1). .

In order to test the utility and generality of the hierarchical methods of the present invention, we designed to build longer genes encoding Pfu DNA polymerase (about 2,325 bp) as a model target. We designed oligonucleotide sequences for the assembly of seven different 400 bp fragments. We anticipated that assembly of overlapping 400 bp fragments could cause interference during the fragment assembly process (FIG. 4). Thus, we designed oligonucleotides that do not overlap one another during the assembly process by introducing different replicative primer sequences for each target fragment of 400 bp. We assembled four 400 bp fragments using a pair of replicable flanking DNA sequences containing Bsg I restriction enzyme sites, and the other three 400 bp fragments contained another Bts I restriction enzyme site. Assembled using pairs of replicable flanking DNA sequences (FIG. 5). After assembling seven target DNA fragments in two separate reaction mixtures, we cleaved the replicable flanking sequences using Bsg I and Bts I restriction enzymes in the reaction mixture, respectively. Finally, we assembled the DNA fragments into a complete 2,325 bp Pfu gene (FIG. 5). To identify our assembly method, we cloned and sequenced the final product. Sequencing obtained from the synthesis of the Pfu gene according to the method of the present invention showed an error rate of about 1 bp per 200 bp compared to the synthesis of the Pfu gene using column-synthesized oligonucleotides (see: Hierarchically synthesized in Table 1). Sequencing data of Pfu DNA polymerase).

In this report, we presented a novel microchip DNA-based gene synthesis method. By applying a two-layer replicable flanking DNA sequence, we achieved a hierarchical assembly method of genes greater than 1 kb using oligonucleotides derived from programmable DNA microchips. The hierarchical strategy of the present invention may be extended to multiple layers of primer sequences for the synthesis of large DNA molecules. Thus, the inventors anticipate that our hierarchical strategy that meets optimized synthetic conditions may be applied to further expand the construction of many synthetic gene sequences. It is also noteworthy that we have achieved one-pot synthesis of genes greater than 1 kb using our optimized assembly process. As mentioned above, the construction of DNA molecules larger than 1 kb was greatly difficult due to the difficulty of DNA assembly associated with very small amounts of chip oligonucleotides.

Error rates and cost estimates for gene synthesis using Agilent chip-oligonucleotides are as follows. In one gene synthesis cost analysis, we found that the two major costs for gene synthesis are the oligonucleotide cost and the sequencing cost. In the 150 bp oligonucleotide of the Agilent 55K chip, two 25 bp flanking sequences are present and 100 bp internal sequences are used for target gene synthesis. Since each 100 bp oligonucleotide perfectly overlaps with another 100 bp oligonucleotide, one oligonucleotide is used for 50 bp extension during the gene assembly process. Thus, we will be able to synthesize 2.75 million bp (50 bp55,000 oligonucleotide) using one 55K chip. Since the price of the Agilent chip is about $ 7,000, we estimate the cost of synthesizing the oligonucleotide to be about $ 0.0025 per bp. Since the current resin-based oligonucleotide synthesis cost is about $ 0.10 per bp, the cost of Agilent chip oligonucleotides is more than 40 times cheaper than the resin-based oligonucleotide synthesis cost.

Sequencing costs are largely determined by the original quality of the oligonucleotides. To assess the quality of Agilent chip oligonucleotides, we amplified and cloned the chip oligonucleotides followed by DNA sequencing. Six errors (5 deletions and one insertion) were observed at 1,500 bp sequencing (ie 1 bp error rate per 250 bp). Using 0.4% error rate oligonucleotides for the production of 1,056 bp of Dpo4 gene would only produce 1.5% of the desired clones containing the complete target DNA (calculated arithmetically from 0.996 ¹⁰⁵⁶ ). During the DNA assembly process, high annealing temperatures allow for the positive selection of higher purity oligonucleotides for expansion of the DNA fragments. As a result, our one-pot Dpo4 synthesis method resulted in an error rate of about 1 bp per 500 bp. In general, we predicted that about 10% of the clones would be error free on average at an error rate of about 1 bp per 500 bp, with about 37% of the single-step Dpo4 gene synthesis obtained in the Dpo4 gene synthesis of the present invention. There were no errors in the clones (3 of 8). Thus, Dop4 gene synthesis using DNA microchip oligonucleotides would cost about $ 2.5 per total oligonucleotide required (calculated: 1,056 bp $ 0.0025 / bp) and total cost of sequencing required would cost about $ 100 (calculated: 10 clones) Sequencing / clone $ 5 / sequencing). Given that the resin-based synthetic oligonucleotide costs about $ 200 (calculated: 21,056 bp $ 0.1 / bp; 2 means forward and reverse strands), chip-based DNA synthesis reduces gene synthesis costs from about $ 300 to about Reduces to $ 100

The error rate of Pfu DNA polymerase gene synthesis was about 2.5 times higher than that of one-pot Dpo4 synthesis. This will result from the additional steps required for hierarchical DNA synthesis. Therefore, we propose that current useful error correction methods (Carr and Church, 2009) should be combined with our DNA synthesis method which can further reduce the error rate of DNA synthesis.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

references

Bang, D., and Church, G.M. (2008). Gene synthesis by circular assembly amplification. Nat Methods 5, 37-39.

Borovkov, A.Y., Loskutov, A.V., Robida, M.D., Day, K.M., Cano, J.A., Le Olson, T., Patel, H., Brown, K., Hunter, P.D., and Sykes, K.F. (2010) High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides. Nucleic Acids Res.

Carr, P.A., and Church, G.M. (2009). Genome engineering. Nat Biotechnol 27, 1151-1162.

Cello, J., Paul, A. V., and Wimmer, E. (2002). Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science 297, 1016-1018.

Coleman, J.R., Papamichail, D., Skiena, S., Futcher, B., Wimmer, E., and Mueller, S. (2008). Virus attenuation by genome-scale changes in codon pair bias. Science 320, 1784-1787.

Czar, M. J., Anderson, J. C., Bader, J. S., and Peccoud, J. (2009). Gene synthesis demystified. Trends Biotechnol 27, 63-72.

Gibson, DG, Benders, GA, Andrews-Pfannkoch, C., Denisova, EA, Baden-Tillson, H., Zaveri, J., Stockwell, TB, Brownley, A., Thomas, DW, Algire, MA, et al . (2008). Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215-1220.

Gibson, DG, Glass, JI, Lartigue, C., Noskov, VN, Chuang, RY, Algire, MA, Benders, GA, Montague, MG, Ma, L., Moodie, MM, et al . (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52-56.

Hutchison, C.A., 3rd, Smith, H.O., Pfannkoch, C., and Venter, J.C. (2005). Cell-free cloning using phi29 DNA polymerase. Proc Natl Acad Sci U S A 102, 17332-17336.

Kodumal, S.J., Patel, K.G., Reid, R., Menzella, H.G., Welch, M., and Santi, D.V. (2004). Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc Natl Acad Sci U S A 101, 15573-15578.

Lee, C. C., Snyder, T. M., and Quake, S. R. (2010) A microfluidic oligonucleotide synthesizer. Nucleic Acids Res 38, 2514-2521.

LeProust, E.M., Peck, B.J., Spirin, K., McCuen, H.B., Moore, B., Namsaraev, E., and Caruthers, M.H. (2010) Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res 38, 2522-2540.

Mueller, S., Coleman, JR, Papamichail, D., Ward, CB, Nimnual, A., Futcher, B., Skiena, S., and Wimmer, E. (2010) Live attenuated influenza virus vaccines by computer-aided rational design. Nat Biotechnol 28, 723-726.

Mueller, S., Coleman, J. R., and Wimmer, E. (2009). Putting synthesis into biology: a viral view of genetic engineering through de novo gene and genome synthesis. Chem Biol 16, 337-347.

Richmond, K.E., Li, M.H., Rodesch, M.J., Patel, M., Lowe, A.M., Kim, C., Chu, L.L., Venkataramaian, N., Flickinger, S.F., Kaysen, J., et al. (2004). Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. Nucleic Acids Res 32, 5011-5018.

Smith, H.O., Hutchison, C.A., 3rd, Pfannkoch, C., and Venter, J.C. (2003). Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci U S A 100, 15440-15445.

Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M., and Heyneker, H. L. (1995). Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49-53.

Tian, J., Gong, H., Sheng, N., Zhou, X., Gulari, E., Gao, X., and Church, G. (2004). Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432, 1050-1054.

Tian, J., Ma, K., and Saaem, I. (2009). Advancing high-throughput gene synthesis technology. Mol Biosyst 5, 714-722.

Tumpey, T.M., Basler, C.F., Aguilar, P.V., Zeng, H., Solorzano, A., Swayne, D.E., Cox, N.J., Katz, J.M., Taubenberger, J.K., Palese, P., et al. (2005). Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310, 77-80.

Welch, M., Govindarajan, S., Ness, J.E., Villalobos, A., Gurney, A., Minshull, J., and Gustafsson, C. (2009). Design parameters to control synthetic gene expression in Escherichia coli. PLoS One 4, e7002.

Zhou, X., Cai, S., Hong, A., You, Q., Yu, P., Sheng, N., Srivannavit, O., Muranjan, S., Rouillard, JM, Xia, Y., et al. (2004). Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences. Nucleic Acids Res 32, 5409-5417.

Claims (9)

Hierarchical preparation of a target nucleic acid sequence comprising the following steps:
(a) 5'-terminal first common priming sequence in the 5 'to 3' direction, primary 5'-restriction cleavage sequence, overlapping target partial sequence, primary 3 ' Preparing an oligonucleotide pool comprising a restriction enzyme cleavage sequence and a 3′-terminal first common priming sequence;
Some of the overlapping target sequences in the oligonucleotide pool comprise the entire target nucleic acid sequence when they overlap each other, and the primary 5′-limiting enzyme cleavage sequence and the primary 3′-limiting enzyme cleavage sequence of the oligonucleotide Are the same as each other;
The first oligonucleotide for generating the 5'-terminal portion of the first intermediate product in the oligonucleotide pool is a 5'-second common priming sequence in the 5 'to 3' direction downstream of the 5'-restriction cleavage sequence. And a second oligonucleotide to further generate a 3'-terminal portion of the first intermediate product, wherein the second oligonucleotide further comprises a 3 'to 5' upstream of the 3'-restriction cleavage sequence. Additionally a 3'-second common priming sequence and a second 3'-restriction cleavage sequence in the direction;
The third oligonucleotide for generating the 5'-terminal portion of the second intermediate product is a 5'-third common priming sequence and a third 5 in the 5 'to 3' direction downstream of the 5'-restriction cleavage sequence. A fourth oligonucleotide comprising a '-restriction cleavage sequence, wherein the fourth oligonucleotide for generating the 3'-terminal portion of the second intermediate product is a 3'-agent upstream of the 3'-restriction cleavage sequence in a 3' to 5 'direction. Further comprises a third common priming sequence and a third 3′-limiting enzyme cleavage sequence;
(b) amplifying the oligonucleotide pool with a polymerase chain reaction (PCR) using a first primer set that hybridizes to the 5'-terminal first common priming sequence and the 3'-terminal first common priming sequence;
(c) cleaving the amplification product of step (b) with a restriction enzyme that acts on the first 5′-limiting enzyme cleavage sequence and the first 3′-limiting enzyme cleavage sequence;
The cleavage causes the 5′-terminal first common priming sequence and 3′-terminal first common priming sequence to be released from the pool into the oligonucleotides;
A second common priming sequence is located at the 5'-end of the first oligonucleotide, a second common priming sequence is located at the 3'-end of the second oligonucleotide, and a 5'-end of the third oligonucleotide. A third common priming sequence is located, and a third common priming sequence is located at the 3′-end of the fourth oligonucleotide;
(d) a first assembly amplification reactant 1 comprising (i) a set of primers hybridizing to a second common priming sequence of the first oligonucleotide and a second common priming sequence of the second oligonucleotide and (ii) the third A first assembly amplification reaction was carried out using a first assembly amplification reactant 2 comprising a primer set hybridized to a third common priming sequence of the oligonucleotide and a third common priming sequence of the fourth oligonucleotide to obtain a first intermediate product. intermediates) and a second intermediate product;
(e) replacing the first intermediate product and the second intermediate product of step (d) with (i) the second 5′-limiting enzyme cleavage sequence of the first oligonucleotide and the second 3 ′ of the second oligonucleotide. A restriction enzyme that acts on the restriction enzyme cleavage sequence and (ii) a third 5'-limiter cleavage sequence of the third oligonucleotide and a third 3'-limiter cleavage sequence of the fourth oligonucleotide Cleaving with restriction enzymes; And
(f) assembling the result of step (e) to obtain a target nucleic acid sequence.
The method of claim 1, wherein the oligonucleotide pool of step (a) is derived from a DNA microchip.
The method of claim 1, wherein the number of oligonucleotides forming the first intermediate product and the second intermediate product of step (a) is at least three.
4. The method of claim 3, wherein the number of oligonucleotides is at least four.
The method of claim 1, wherein the second restriction enzyme cleavage sequence and the third restriction enzyme cleavage sequence of step (a) are different from each other.
The method of claim 1, wherein the amplification of the first intermediate product and the second intermediate product of step (b) is performed in different tubes.
The method of claim 1, wherein the size of the first intermediate product and the second intermediate product of step (b) is 300-500 bp.
The method of claim 1, wherein the assembly PCR carried out in the method is carried out at an annealing temperature of 65 ℃ or more hierarchical manufacturing method.
delete

Images