CN107250446B

CN107250446B - Oligonucleotides and uses thereof

Info

Publication number: CN107250446B
Application number: CN201580075708.6A
Authority: CN
Inventors: 康康; 陈世宏; 沈玥; 王云; 徐讯; 田埂
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2015-06-11
Filing date: 2015-06-11
Publication date: 2020-04-14
Anticipated expiration: 2035-06-11
Also published as: CN107250446A; WO2016197374A1

Abstract

An oligonucleotide and application thereof. The oligonucleotides include: a gene coding region; the kit comprises a first flanking sequence formed at the 5 'end of a gene coding region and a second flanking sequence formed at the 3' end of the gene coding region, wherein a plate library sorting primer binding sequence, a component sub library sorting primer binding sequence and an enzyme cutting site are sequentially formed on the first flanking sequence from the 5 'end to the 3' end, and an enzyme cutting site, a component sub library sorting primer binding sequence and a plate library sorting primer binding sequence are sequentially formed on the second flanking sequence from the 5 'end to the 3' end.

Description

Oligonucleotides and uses thereof

Technical Field

The invention relates to the field of biotechnology, in particular to oligonucleotide and application thereof.

Background

With the development of genetic science and genetic engineering, DNA synthesis technology plays an increasingly important role in the field of life science. De novo synthesis of DNA elements, including regulatory sequences, complete genes, artificial metabolic pathways, and even complete artificial genomes, will revolutionize human life science research. Since the first synthesis of oligonucleotide chains by humans in 1961, techniques for DNA synthesis and assembly have been developed. Currently, the synthesis and assembly of long DNA fragments are assembled by oligonucleotide chains of 100bp or less due to technical limitations, and the cost is approximately stabilized at about 2.2 RMB per bp. Therefore, the conventional methods for DNA synthesis and assembly, which are cost-prohibitive, are difficult to apply to genomic DNA synthesis.

In 2004, Jingdong et al successfully synthesized 292 different oligonucleotide chains on a DNA chip and assembled them into 14.6kb DNA fragments using an oligonucleotide microarray chip, enabling efficient, low-cost, large-scale DNA long-fragment synthesis and assembly based on chip technology (Tian (2004) Nature 432: 1050). In 2010, Kosuri et al achieved the synthesis of hundreds of genes using commercial DNA microarray chips from agilent (kosuret al (2010) nat. biotech.28:1295) and provided a complete and complete technical route (eroschenkoe et al (2012) curr. protocol. in chem. biol.4: 1). Based on this related art, in 2012, GEN9 company established in the united states as the first commercial company providing DNA synthesis services worldwide, and its product price for DNA synthesis is about $ 0.26 per bp, which is lower than the market price for traditional DNA synthesis.

Two major challenges facing DNA synthesis technology are the high error rate of the oligonucleotide strands and the impact of the high complexity of the oligonucleotide library on assembly. How to further improve the synthesis accuracy of the DNA chip synthesis technology and reduce the assembly and screening costs plays a crucial role in the commercial application of the DNA chip synthesis technology.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. To this end, an object of the present invention is to propose an oligonucleotide that can be effectively used for DNA synthesis and its applications.

The present invention has been completed based on the following findings of the inventors:

the longer the synthesis length of the oligonucleotide library synthesized on the DNA microarray chip, the lower the accuracy. At present, Agilent, a chip technology company, can provide an oligonucleotide library within 200nt, and most chip manufacturers recommend that the length of the oligonucleotide be controlled within 60nt or 120nt in order to ensure the product quality. According to the technical scheme provided by Kosuri et al (see FIG. 10 in the technical scheme), redundant flanking sequences are reserved at both ends of the oligonucleotide library for oligonucleotide screening of different assembly reactions, including 40nt primer binding sequences and 6nt cleavage site sequences, i.e., about 92nt of each oligonucleotide strand is not involved in DNA assembly. When the length of the oligonucleotide library is limited to 120nt, the effective synthesis length is only 28nt at the maximum, which is not enough to complete DNA assembly by bridge PCR; when the oligonucleotide library is limited to 160nt, the effective synthesis length is 68nt, and DNA assembly can be barely carried out. The shorter the effective synthesis length of the oligonucleotide chain, the more oligonucleotide chain species are involved in the assembly of each DNA fragment, and the assembly success rate is greatly reduced. Therefore, the method has obvious practical significance for reducing the length of flanking sequences of the oligonucleotide library, increasing the effective assembly length, further reducing the complexity of the oligonucleotide library and the complexity of an assembly mixture, greatly reducing the DNA synthesis cost and improving the power of synthesis and assembly.

Based on the above situation, the present invention aims to provide a design scheme of Oligonucleotide Library flanking primers for DNA Synthesis technology, so as to effectively increase the effective fragment length of Oligonucleotide chains on a DNA microarray chip when a large fragment of nucleic acid sequence is synthesized by Oligonucleotide Library Synthesis (OLS) strategy, thereby increasing the carrying amount of the chip on the synthesized sequence, i.e. one chip can synthesize more sequences, and further significantly reduce the Synthesis cost.

In a first aspect of the invention, the invention provides an oligonucleotide. According to an embodiment of the invention, the oligonucleotide comprises: a gene coding region; a first flanking sequence formed 5' of the coding region of the gene; and a second flanking sequence, wherein the second flanking sequence is formed at the 3' end of the gene coding region, the first flanking sequence sequentially forms a plate bank sorting primer binding sequence, a component sub-bank sorting primer binding sequence and an enzyme cutting site from the 5' end to the 3' end, the second flanking sequence sequentially forms an enzyme cutting site, a component sub-bank sorting primer binding sequence and a plate bank sorting primer binding sequence from the 5' end to the 3' end, and the plate bank sorting primer binding sequence, the component sub-bank sorting primer binding sequence and the enzyme cutting site meet at least one of the following conditions: (a) the plate bank sorting primer binding sequence and the component bank sorting primer binding sequence are public primer binding sequences; (b) at least one part of the enzyme cutting sites are formed inside the sorting primer binding sequences of the component sub-library, and the enzyme cutting sites are connected with the gene coding regions; (c) the plate sub-library sorting primer binding sequence shares a partial overlap sequence with the component sub-library sorting primer binding sequence. The inventor surprisingly finds that the oligonucleotide can be effectively used for DNA synthesis, and the effective segment length of the oligonucleotide can be effectively increased and the assembly difficulty can be greatly reduced by using the oligonucleotide for DNA synthesis, so that the carrying amount of the synthetic DNA of an oligonucleotide library can be greatly increased, and the DNA synthesis cost can be further reduced. According to the embodiment of the invention, the oligonucleotide can effectively reduce the DNA synthesis cost by 40-50%, greatly improve the flux of DNA products synthesized by each chip, and in addition, the primer synthesis cost can be reduced by about 50% by using a common primer.

According to an embodiment of the invention, the oligonucleotide is 100 to 300nt in length.

According to an embodiment of the invention, the cleavage site is 6-12nt in length.

According to an embodiment of the present invention, the cleavage site is a type II restriction enzyme cleavage site.

According to an embodiment of the invention, the length of the common primer binding sequence, the plate subpool sorting primer binding sequence, the component subpool sorting primer binding sequence is 15-25 nt.

According to an embodiment of the invention, the common primer binding sequence is a complementary sequence selected from SEQ ID No. 2825.

According to an embodiment of the invention, the plate library sorting primer binding sequence is a complementary sequence of at least one of SEQ ID No. 2815-SEQ ID No. 2824.

According to an embodiment of the invention, the modular sublibrary sorting primer binding sequence is a complementary sequence of at least one of SEQ ID No.1 to SEQ ID No. 2814.

According to an embodiment of the invention, the length of the overlapping sequence is not more than 10 nt.

In a second aspect of the invention, the invention provides the use of an oligonucleotide as hereinbefore described in DNA synthesis. The oligonucleotide is utilized to carry out DNA synthesis, the effective segment length of the oligonucleotide can be effectively increased, and the assembly difficulty is greatly reduced, so that the synthetic flux of an oligonucleotide library is greatly improved, and the synthetic cost is further reduced. According to the embodiment of the invention, the oligonucleotide can effectively reduce the DNA synthesis cost by 40-50%, greatly improve the flux of DNA products synthesized by each chip, and in addition, the primer synthesis cost can be reduced by about 50% by using a common primer.

In a third aspect of the invention, the invention provides a method of synthesizing DNA. According to an embodiment of the invention, the method comprises: synthesizing an oligonucleotide library using the DNA chip; and assembling based on the oligonucleotide library to obtain the target DNA, wherein the oligonucleotide library comprises a plurality of oligonucleotide chains, the oligonucleotide chains are the oligonucleotides, and specifically, the assembling based on the oligonucleotide library means that the plurality of oligonucleotide chains are subjected to enzyme digestion to cut off the primer binding sites to obtain DNA fragments, and then the obtained DNA fragments are assembled to obtain the target DNA. The method of the invention can be used for quickly and effectively synthesizing DNA, the effective segment length of the oligonucleotide is longer, the assembly difficulty is lower, and the cost of DNA synthesis can be reduced in a large scale. According to one embodiment of the invention, the method can effectively reduce the DNA synthesis cost by 40-50%, greatly improve the flux of DNA products synthesized by each chip, and in addition, the primer synthesis cost can be reduced by about 50% by using the common primer.

In a fourth aspect of the present invention, the present invention provides a DNA. According to an embodiment of the present invention, the DNA is synthesized by the method for synthesizing DNA described above. It should be noted that all the features and advantages of the oligonucleotide and the method for synthesizing DNA described above are applicable to the DNA and are not described in detail herein.

Drawings

FIG. 1 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 2 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 3 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 4 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 5 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 6 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 7 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 8 shows a schematic diagram of the structure of an oligonucleotide chain according to one embodiment of the present invention;

FIG. 9 shows an electropherogram of successfully assembled DNA modules, according to one embodiment of the invention; and

FIG. 10 is a schematic diagram showing a process flow of a method for synthesizing DNA according to an embodiment of the present invention, wherein the left diagram is a DNA synthesis experiment operation flow, the right diagram is a schematic diagram of an oligonucleotide reaction, the horizontal line represents an oligonucleotide (single strand) or a DNA small molecule (double strand), the vertical line indicates the position of a sorting primer, F represents a plate library sorting primer of an F plate library, and 2 represents a module library sorting primer of module No.2 in the F plate library.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In a first aspect of the invention, the invention provides an oligonucleotide. According to an embodiment of the present invention, referring to fig. 1, the oligonucleotide includes: a gene coding region; a first flanking sequence formed 5' of the coding region of the gene; and a second flanking sequence formed 3' of the coding region of the gene. Wherein, a plate bank sorting primer binding sequence, a component sub-bank sorting primer binding sequence and an enzyme cutting site are sequentially formed on the first flanking sequence from a 5 'end to a 3' end, and an enzyme cutting site, a component sub-bank sorting primer binding sequence and a plate bank sorting primer binding sequence are sequentially formed on the second flanking sequence from a 5 'end to a 3' end, wherein the plate bank sorting primer binding sequence, the component sub-bank sorting primer binding sequence and the enzyme cutting site satisfy at least one of the following and combinations thereof: (a) the plate bank sorting primer binding sequence and the component bank sorting primer binding sequence are common primer binding sequences (namely, the plate bank sorting primer binding sequence and the component bank sorting primer binding sequence are replaced by the common primer binding sequence); (b) at least one part of the enzyme cutting sites are formed inside the sorting primer binding sequences of the component sub-library, and the enzyme cutting sites are connected with the gene coding regions; (c) the plate sub-library sorting primer binding sequence shares a partial overlap sequence with the component sub-library sorting primer binding sequence. The inventor surprisingly finds that the oligonucleotide can be effectively used for DNA synthesis, and the effective segment length of the oligonucleotide can be effectively increased and the assembly difficulty can be greatly reduced by using the oligonucleotide for DNA synthesis, so that the carrying amount of the synthetic DNA of an oligonucleotide library can be greatly increased, and the DNA synthesis cost can be further reduced. According to the embodiment of the invention, the oligonucleotide can effectively reduce the DNA synthesis cost by 40-50%, greatly improve the flux of DNA products synthesized by each chip, and in addition, the primer synthesis cost can be reduced by about 50% by using a common primer.

According to the embodiment of the invention, the plate bank sorting primer binding sequence, the component bank sorting primer binding sequence and the enzyme cutting site in the first flanking sequence and the second flanking sequence are not affected with each other, and the specific sequences may be the same or different.

According to an embodiment of the present invention, the cleavage site is a type II restriction enzyme cleavage site, for example, a Btsl cleavage site.

According to an embodiment of the invention, the common primer binding sequence may be the complement of SEQ ID No. 2825.

According to an embodiment of the invention, the modular sublibrary sorting primer binding sequence is a complementary sequence of at least one of SEQ ID No.1 to SEQ ID No. 2814. Wherein, the sequences shown by SEQ ID NO. 1-2800 are shown in Table 1.

TABLE 1

According to an embodiment of the invention, the length of the overlapping sequence is not more than 10 nt. This is advantageous in maintaining the specificity of the sorting primer.

According to a specific example of the present invention, referring to fig. 2, the plate bank sorting primer binding sequence and the component bank sorting primer binding sequence satisfy the above condition (a), that is, the plate bank sorting primer binding sequence and the component bank sorting primer binding sequence are replaced with a common primer binding sequence. Therefore, the primer binding sequence with one end of 40nt is reduced into the common primer with 15-25nt, on one hand, the synthesis cost of the primer is reduced by nearly 50%, and on the other hand, the effective synthesis and extension length are prolonged by 15-25 nt. However, it should be noted that the common primer does not have a sorting function, and is used in combination with a slat sub-library sorting primer and an assembly sub-library sorting primer in the sorting process of the plate sub-library and the assembly sub-library to realize sorting. Since the 5 'end is of higher quality and the 3' end is of lower quality when the oligonucleotide is synthesized, it is recommended that the portion of lower quality fall as little as possible into the effective synthetic fragment requiring fidelity and relatively as much as possible into the flanking sequence, and therefore it is preferable to use a common primer at the 5 'end of the oligonucleotide strand and two sort primers at the 3' end, respectively.

According to a specific example of the present invention, referring to fig. 3, the enzyme cutting site and the assembly sub-library sorting primer binding sequence satisfy the condition (b) that at least a part of the enzyme cutting site is formed inside the assembly sub-library sorting primer binding sequence, and the enzyme cutting site is connected with the gene coding region. Thus, placing one end restriction endonuclease site inside the modular sublibrary sorting primer will effectively synthesize and extend a length by 6-12 nt. Since the 3 'end sequence of the primers determines the specificity of the PCR reaction, the use of a consensus cleavage site as the 3' end of the primers will reduce the specificity of the PCR to some extent, it is preferable not to use this optimization at both ends of the oligonucleotide library to avoid non-specific amplification. It should be noted that, in this example, since restriction enzyme sites are placed in the primer recognition sites, the versatility is reduced, and it is not recommended to synthesize a sequence containing such restriction enzyme sites.

According to a specific example of the present invention, referring to fig. 4, the plate bank sorting primer binding sequence and the component sub-bank sorting primer binding sequence satisfy the above condition (c), i.e., the plate bank sorting primer binding sequence shares a partially overlapping sequence with the component sub-bank sorting primer binding sequence. Thus, the modular subsublibrary sorting primer binding sequence shares a sequence (e.g., can be 10nt) with the plate library sorting primer binding sequence, which increases the effective synthesis and extension length by twice the length of the overlap region (e.g., 20 nt). In addition, to ensure that the two pairs of sorting primers retain their respective specificities, it is preferable that the overlapping region is not longer than 10 nt.

According to a specific example of the present invention, referring to fig. 5, the enzyme cleavage site, the plate subpool sorting primer binding sequence and the module subpool sorting primer binding sequence satisfy the above conditions (a) and (b) at the same time. Thus, the effective synthesis and extension length can be increased by 21-37 nt.

According to a specific example of the present invention, referring to fig. 6, the plate bank sorting primer binding sequence and the component bank sorting primer binding sequence satisfy both of the above conditions (a) and (c). Therefore, the effective synthesis and extension length can be increased by 15-35nt, the assembly difficulty of DNA synthesis is effectively reduced, and the cost is greatly reduced.

According to a specific example of the present invention, referring to fig. 7, the cleavage site, the plate subpool sorting primer binding sequence and the module subpool sorting primer binding sequence satisfy the above conditions (b) and (c) at the same time. Therefore, the effective synthesis and extension length can be increased by 6-32nt, the assembly difficulty of DNA synthesis is effectively reduced, and the cost is greatly reduced.

According to a specific example of the present invention, referring to fig. 8, the enzyme cleavage site, the plate subpool sorting primer binding sequence and the module subpool sorting primer binding sequence satisfy the above conditions (a), (b) and (c) at the same time. The effective synthesis and extension length can be increased by 21-47 nt. The inventor surprisingly finds that the oligonucleotide can be effectively used for DNA synthesis, and the effective segment length of the oligonucleotide can be effectively increased and the assembly difficulty can be greatly reduced by using the oligonucleotide for DNA synthesis, so that the flux of a DNA chip can be greatly improved and the DNA synthesis cost can be reduced. According to the embodiment of the invention, the oligonucleotide can effectively reduce the DNA synthesis cost by 40-50%, greatly improve the flux of DNA products synthesized by each chip, and in addition, the primer synthesis cost can be reduced by about 50% by using a common primer.

In a second aspect of the invention, the invention provides the use of an oligonucleotide as hereinbefore described in DNA synthesis. The oligonucleotide provided by the invention is used for DNA synthesis, so that the effective fragment length of the oligonucleotide can be effectively increased, the assembly difficulty is greatly reduced, and the cost for synthesizing an oligonucleotide library is reduced on a large scale. According to the embodiment of the invention, the oligonucleotide can effectively reduce the DNA synthesis cost by 40-50%, greatly improve the flux of DNA products synthesized by each chip, and in addition, the primer synthesis cost can be reduced by about 50% by using a common primer.

To verify the effectiveness of the present invention, partial sequences in the artificial chromosome 2 and chromosome 7 of the SC #2.0 project (artificial yeast genome) were used for verification. All DNA components are artificially designed yeast genome fragments, and the length is 450-650 bp.

The sequence information of the 12 DNA modules and the corresponding primer sequence information and oligonucleotide (oligos) library sequence information designed according to different protocols are listed below. Synthesizing oligos on DNA chip according to designed sequence, amplifying and assembling according to corresponding primer, and completing test.

The experimental procedures in the following examples are described in the literature by Nikolai Eroshenko et al (Gene Assembly from Chip-Synthesized Oligonucleotides, Nikolai Eroshenko, Current Protocols in chemical Biology4:1-17, March 2012) (see FIG. 10 for a flow chart). The scheme adopted by the invention comprises the following steps and combinations thereof:

(a) the plate bank sorting primer binding sequence and the component bank sorting primer binding sequence are public primer binding sequences;

(b) at least one part of enzyme cutting sites are formed inside the sorting primer binding sequence of the assembly sublibrary, and the enzyme cutting sites are connected with the gene coding region;

(c) the plate bank sorting primer binding sequence shares a partial overlap sequence with the component bank sorting primer binding sequence.

Example 1

Name of DNA Module: BBK515

The experimental scheme is as follows: the original protocol (i.e., the prior art protocol disclosed in the literature by Nikolai Eroshenko) was used as a control for the present invention.

Length of DNA module: 581bp

DNA module sequence:

GGTCTCCTCGAGTACAGCTATCTTGGTTCAGACGTGAGAAGTGATGCACAGCAGCTGTATCCTTTATTTATTGGAGCTTCCGAAACTTCCCAAGAAAAGTGAAGAAAAAACCAGGAAAATCAAAAGACATGACGCATATAATGTACACCTTTTTTTTTTCGCGGCCAAAAATTTAACCTACTCATCTAGATCAGTGGTTGGTAGAGCAGTATTCCGTCATTTTTCATGTCCAGGTTCGAAGCTGACCTGGTATTATCCGGTTTTTTTTCTTGTTGACCAATAGGAAAAAAAAAAATTAAAATAAAGTCACAGAAATTCAATGAATATTATAAATTTTTCTTGTTTGTTTCCCTAGTTGTTATTTTTATAAAAAAAATTCTTGTAGACAATAAAATAAGAAATGCCCATTTTGTAACTTAGCGAAAGATGCCCAGTACATCCCTTTTACACCCGTGCATTAAAGGTGTTTGGGTTTAATAGGAGCTTTATCATATCTCTTTGATTTTTTTTCTGCTGTCCTCGGCTTGAGGGACTCACAGAGATCTGGAAATTTTCAGATTGTCAGTGCTTAGGATGAGACC

plate library sorting primers:

skpp-5-F TCCGACGGGGAGTATATACT(SEQ ID NO.2815)

skpp-5-R TACTAACTGCTTCAGGCCAA(SEQ ID NO.2816)

sorting primers of the component sub-library:

skpp-515-F ATAGTGCTGTAGTGGAACCG(SEQ ID NO.2801)

skpp-515-R TTCTTACTAGAGACTGCGCC(SEQ ID NO.2802)

assembling a primer:

skpp-115-F GAGCCATGTGAAATGTGTGT(SEQ ID NO.2826)

skpp-115-R CGGACTAAAGGATCGAGTCA(SEQ ID NO.2827)

example 2

Name of DNA Module: BBK516

Length of DNA module: 578bp

DNA module sequence:

GGTCTCTAGGATGGGTTGTCAGTAGACGGTGGCCGCCGTGGATGGGAAATCTCATACGTTTACACACATAGTGTTTGGAAATTAATAGTAGCAATAGCTATCTGGCTACTGTTTTAAAGTATTAGCCCGTTCTCAGTGCTTCTTTTTTAAGGAATAACAACGGCAAGACCAAAGATATATCAAATATGGCTAAGCAATCTCTAGGTATGTTTGGAGGATACGAATAACGATAGAAAACATGAGTGAATTTCCGTCCACGAAAAAATGTTAACATAAAATGCAAGAGAACAATTAATCGAATAATGTTAAATTATTGTAAAACAATGTGTATGATGAGGAGGAATGTACCTAAGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAACAGCTTTTGCATATTCAATCCAGGCATAGGGCGACTATTTAGCACTCAACGATTTTTAAGCTTGTGTATTGCTGACATAAATTCCGGCTTTAGAATCCAATATTGAAAAACGTGAGTACGCAGAGGAGATAGCGCGCCAGCACTACATCAACTGACTACTGACTACTGACTGCCACCTCGAGGAGACC

plate library sorting primers:

skpp-5-F TCCGACGGGGAGTATATACT(SEQ ID NO.2815)

skpp-5-R TACTAACTGCTTCAGGCCAA(SEQ ID NO.2816)

sorting primers of the component sub-library:

skpp-516-F CGTGTAGTGTGAATATGCGG(SEQ ID NO.2803)

skpp-516-R GAGTCAATGATTGAGCCTGC(SEQ ID NO.2804)

assembling a primer:

skpp-116-F CGTATACGTAAGGGTTCCGA(SEQ ID NO.2828)

skpp-116-R CATCGGATAACACAAAGCGT(SEQ ID NO.2829)

example 3

Name of DNA Module: BBK802

The experimental scheme is as follows: a is

Length of DNA module: 579bp

DNA module sequence:

GGTCTCAAGAAGGCGTTCTTCCAGCATAATTTCACATGTGGAACCGGAGACTTTTGAAGATGAAAATGACCAGCAACTTCTACCAAATATGAATGCTACTTGGGTAGACCAACGCGGCGCTTGGATTATTCATGTGGTCATTATCATACTGCTGAAACTATTTTATAATTTATTTCCTGGCGTCACTACCGAATGGAGCTGGACCTTAACTAATATGACATATGTTATTGGGTCCTATGTCATGTTCCATCTGATTAAGGGTACCCCTTTCGATTTCAATGGTGGTGCTTATGACAACTTGACGATGTGGGAACAAATTGACGACGAGACTTTATATACTCCTTCAAGAAAATTTTTGATTAGTGTCCCGATCGCCCTATTCTTAGTTAGTACTCATTATGCTCACTATGATTTGAAATTGTTTTCATGGAATTGTTTTTTGACAACCTTTGGTGCTGTTGTCCCAAAGTTACCTGTTACTCATAGATTAAGGATTTCTATCCCTGGCATTACCGGCAGAGCTCAAATCTCATGAACAATAACTTCGTATAATGTACATTATACGAAGTTATTGAGACC

5' common primer

skpp-1-F ATATAGATGCCGTCCTAGCG(SEQ ID NO.2825)

Plate library sorting primers:

skpp-8-R ATGATCCTATGCGTCTGTGT(SEQ ID NO.2817)

sorting primers of the component sub-library:

skpp-801-R GAACCATTCCTTTGCTGACC(SEQ ID NO.2805)

assembling a primer:

skpp-102-F TTTGCTTCAGTCAGATTCGC(SEQ ID NO.2830)

skpp-102-R GTTCAATCACTGAATCCCGG(SEQ ID NO.2831)

example 4

Name of DNA Module: BBK803

The experimental scheme is as follows: a is

Length of DNA module: 595bp

DNA module sequence:

GGTCTCGTTATTAGTTTATTTCAATCTTGTATCTAGATGTTTTTGCTATATTTATATTTTCTGGGCCAAATCACTAACGCTTTGAAATTTCAAAAGCCAACAACGGATTTCGGACTTCATTTATGTAAATGAAAAAGAATGTGAACAAAAATTGAAAAAATTCCATATTAATGCACCTTCTAAACCTATTTGTTGAAATTGTATTTAATTCATATAATCATATTCTATTCAAAAAGAATAACAGATAGTATTAGCACTATTTCACGTACTGCTTCTAATACTATTATCACAAAATAGACATCGGAAAACTCAAAAATAAATAACTTCGTATAATGTACATTATACGAAGTTATATAACTTCGTATAATGTACATTATACGAAGTTATGTATTTATGTTTTGTCATTCTTTTCTACATAATCTTGAAACTAGGTAGATCTACAATTGAAAAGTAAATACTAACATTATTTACTAAATTTAAGTTAGAAATCGGCACGAAAAAAATTTGACAGATTACGAGAGTCCAGCCAAAATATGAGTATATTACTATTTCCCCTTGGTGAAAGAAATGAAAGATGTTATTTTTTACCGAGACC

5' common primer

skpp-1-F ATATAGATGCCGTCCTAGCG(SEQ ID NO.2825)

Plate library sorting primers:

skpp-8-R ATGATCCTATGCGTCTGTGT(SEQ ID NO.2817)

sorting primers of the component sub-library:

skpp-803-R ACTGTGTTAGCCTCGTTTCA(SEQ ID NO.2806)

assembling a primer:

skpp-103-F GTCGAGTCCTATGTAACCGT(SEQ ID NO.2832)

skpp-103-R CAGGGGTCGTCATATCTTCA(SEQ ID NO.2833)

example 5

Name of DNA Module: BBK701

The experimental scheme is as follows: b

Length of DNA module: 632bp

DNA module sequence:

GGTCTCGGATCCTTCTTTTGCACGATGTCACATACTTTTTCGATTACCGTCGCTTTACACAGTAACTGTTTAAAACGAGCGTTCTTAATTAATAACTGTAGGAGAGCAGGCTTTCGGCTTTCTTTATGAGAAATAAGGACTACTCCAGCTTGCCAAGTTTTATCCAGTTACACAAACGCTCAAGATCGCCTCGCTAAAATAGATGAAAAAATAAGAAAAATGAAATGTTCATAACTTCGTATAATGTACATTATACGAAGTTATAACTTAATCCCTTTTGGATTTATCATTGAAAGATCGCTGATTTATTCTTTTCCAATACGCCTCGTAATTCTCTTTCAATGTCAAAGGCGCCAGATTGTAAGAATCTGTCATGTAAAATAATCTGACAATACGTAGTTTTAAAAACCAATTGATTAAACCAAGCTCTCTTATGATGCCAACTATAAAGAAGTACAAGCTACCGGTTAAAGCACCAACGCTGAATCCCACGATTACTTGATCTAAATTGTGGTAGTGCAAGTAAACTCTGGAAAAGCAAACGCAAAACGATAATAAAGCCAATGCACCAGAAAAAATGCATTTTTCTAAGAAGTTTAGATTCTTCCAGGAAGTGTATATCTTCAGAGACC

plate library sorting primers:

skpp-7-F GTACATGAAACGATGGACGG(SEQ ID NO.2818)

skpp-7-R CTGGTATAGTCTCCTCAGCG(SEQ ID NO.2819)

sorting primers of the component sub-library:

skpp-701-IN-F GATTGAAACGGTGAGCAGTG(SEQ ID NO.2807)

skpp-701-R CGTCTATGTTTGATCGACGC(SEQ ID NO.2808)

assembling a primer:

skpp-101-F GCTTATTCGTGCCGTGTTAT(SEQ ID NO.2834)

skpp-101-R TACTTTTGATTGCTGTGCCC(SEQ ID NO.2835)

example 6

Name of DNA Module: BBK702

The experimental scheme is as follows: b

Length of DNA module: 631bp

DNA module sequence:

GGTCTCTCTTCAGAGAGTTATAGGTAAAACAAAACCCCATGAATTGGGAGTGTGCACTGGGCATCCCGTAACCGGATCTTATAGTGTCATTTTGGAACGACGCACCGAACGATACGGGGCGTGGCTGTTTTATTATATTCTTGATCACGTTATTGAATATTTCGTTCATCAATTGGCCAAAAGCAACGATACAAGCTTCCAACTCACGGGTGATGATAAACCACGACAAATAAAAAGCTAGCACTAGGATGGGCATCAGCGAGAAATATGCACTAAGGAATGATAGAAAGTCATGCGAATCATAGAGAATGTATGTGTCATCGAATGGTATAACATTTGGATTTGGATTTATTGCAGCGGCGGTACTATTCATGATATGATCCGATTCAAAACAAATTTTGATGTAGATAAAATGCCTGGTATAATGAGTGGTAGAGAAGAAAATTAGACGAAAAGAATGAGAACGAAGAAATGATTTAAAGTGATCACAGTGGCAATCACTTTCCCCTTTTTAACGCGTGTTGAGCAATCGATTGATGTCTCTACCATATGTAGCATTTTCTCAGAAGGCAAACTTTTCTTTTTCTTTCTACTTTTCCAACAAGAAATTTTAAGTTTTCCCAGAGAGACC

plate library sorting primers:

skpp-7-F GTACATGAAACGATGGACGG(SEQ ID NO.2818)

skpp-7-R CTGGTATAGTCTCCTCAGCG(SEQ ID NO.2819)

sorting primers of the component sub-library:

skpp-702-IN-F GTTCCGTTAATTCGGCAGTG(SEQ ID NO.2809)

skpp-702-R TCAGAAGAAGCGGGAGAATG(SEQ ID NO.2810)

assembling a primer:

skpp-102-F TTTGCTTCAGTCAGATTCGC(SEQ ID NO.2830)

skpp-102-R GTTCAATCACTGAATCCCGG(SEQ ID NO.2831)

example 7

Name of DNA Module: BBK902

The experimental scheme is as follows: c. C

Length of DNA module: 523bp

DNA module sequence:

GGTCTCAATCAAAGAGGCGATATCAACACCTTTTATCCAGCACTATTCAACAGTGAATGGGCTCCCAAGTAAGTCTTGGCATTGTGCTTTCTATTCTTAAGTATTAAGTAGAAGTTTTGTTTACTGGGTTTGTTTATTCCTGGCTAGATGTTCGCATTCGTTTTCTAGTTGACCATATTTACCAAATATTCACAACTAATACCCAGCCAAGGTAGTCTAAAAGCTAATTTCTCTAAAAGGGAGAAAGTTGGTGATTTTTTATCTCGCATTATTATATATGCAAGAATAGTTAAGGTATAGTTATAAAGTTTTATCTTAATTGCCACATACGTACATTGACACGTAGAAGGACTCCATTATTTTTTTCATTCTAGCATACTATTATTCCTTGTAACGTCCCAGAGTATTCCATTTAATTGTCCTCCATTTCTTAACGGTGACGAAGGATCACCATACAACAACTACTAAAGATTATAGTACACTCTCACCTTGCAACTATTTATCTGACATTTGCCTTGAGACC

plate library sorting primers:

skpp-9-F GGCGAGAGGAGATATAGAGC(SEQ ID NO.2820)

skpp-9-R AATATCGAACAGCTGTGCAC(SEQ ID NO.2821)

overlapping element sublibrary sorting primers:

skpp-9-902-F GATATAGAGCCTCCGATTCG(SEQ ID NO.2811)

skpp-9-902-R AGCTGTGCACGTGTTCCGGC(SEQ ID NO.703)

assembling a primer:

skpp-102-F TTTGCTTCAGTCAGATTCGC(SEQ ID NO.2830)

skpp-102-R GTTCAATCACTGAATCCCGG(SEQ ID NO.2831)

example 8

Name of DNA Module: BBK903

The experimental scheme is as follows: c. C

Length of DNA module: 526bp

DNA module sequence:

GGTCTCTGCCTTACTTTTATCTCCAGCTTCCCCTCGATTTTATTTTTCAATTTGATTTCTAAAGCTTTTTGCTTAGGCATACCAAACCATCCACTCATTTAACACCTTATTTTTTTTTTCGAAGACAGCATCCAACTTTATACGTTCACTACCTTTTTTTTTACAACAATTTCATTCTTCATCCTATGAAATGACGAAAATAACCAGAGATGTTTCGATAACGACAGAAAATTCAAAATCCACATCTGGATCGGCAACGGCCTCTTCGGCCTCTTTACCTGAGAACGACCACCCAATATTCCATCAACCTAGAGCTCGTATCCGTAGCGGCAGCTTATTCATCGAAGGATCTGATTCATTTCCATCATCAGAAGTGAAGTCATACAATGTTTATATTGATGATAGCAAGTATAGTGAAATCCTCAAAGGAGATACAAATTCAAGTAGTACTGATGGTAAACAAGTCTTTGAAGATGCTAGAGATGACAATTTCCATCAGGAATCACATAGAGATCTAGAGGAGACC

plate library sorting primers:

skpp-9-F GGCGAGAGGAGATATAGAGC(SEQ ID NO.2820)

skpp-9-R AATATCGAACAGCTGTGCAC(SEQ ID NO.2821)

overlapping element sublibrary sorting primers:

skpp-9-903-F GATATAGAGCGTTTTGGTCG(SEQ ID NO.2812)

skpp-9-903-R AGCTGTGCACGATCTTATAC(SEQ ID NO.703)

assembling a primer:

skpp-103-F GTCGAGTCCTATGTAACCGT(SEQ ID NO.2832)

skpp-103-R CAGGGGTCGTCATATCTTCA(SEQ ID NO.2833)

example 9

Name of DNA Module: BBK1001

The experimental scheme is as follows: a + c

Length of DNA module: 567bp by

DNA module sequence:

CGTCTCCTCGAGTACAGCTATCTTGGTTCAGACGTGAGAAGTGATGCACAGGTCGACTTTTCTTCACTTTTCTTTCAACAATTCAAAGATGGCTAGAACCATAACTTTTGATATCCCTTCCCAATATAAACTCGTAGATTTAATAGGTGAGGGAGCGTACGGAACAGTATGTTCAGCAATTCATAAGCCTTCCGGCATAAAGGTAGCTATCAAGAAAATACAACCGTTTAGCAAAAAATTGTTTGTTACAAGAACTATACGTGAGATCAAGCTTTTACGGTATTTCCATGAACACGAAAACATAATAAGTATATTGGATAAAGTAAGGCCAGTATCCATAGACAAACTAAACGCTGTTTATTTAGTCGAAGAGTTGATGGAAACCGATTTACAAAAAGTAATTAATAATCAGAATAGCGGGTTTTCCACTTTAAGTGATGACCATGTTCAATACTTTACATACCAAATCCTCAGAGCCTTAAAGTCTATTCACAGTGCACAAGTTATCCATAGAGACATAAAGCCATCAAACCTGTTACTAAATTCCAATTGTGATCTCAAGAGACG

5' end common primer:

skpp-1-F ATATAGATGCCGTCCTAGCG(SEQ ID NO.2825)

plate library sorting primers:

skpp-10-R ATATATGGTCACACGCCTGT(SEQ ID NO.2822)

overlapping element sublibrary sorting primers:

skpp-10-1001-R ACACGCCTGTCTATGTCCGC(SEQ ID NO.802)

assembling a primer:

skpp-101-F GCTTATTCGTGCCGTGTTAT(SEQ ID NO.2834)

skpp-101-R TACTTTTGATTGCTGTGCCC(SEQ ID NO.2835)

example 10

Name of DNA Module: BBK1002

The experimental scheme is as follows: a + c

Length of DNA module: 563bp

DNA module sequence:

CGTCTCTCTCAAAGTCTGCGATTTTGGACTAGCTAGATGCTTGGCCTCATCATCAGACAGCAGAGAAACATTGGTAGGATTCATGACGGAGTACGTCGCAACGCGATGGTACAGGGCACCCGAGATAATGCTAACTTTTCAAGAGTACACAACTGCGATGGATATATGGTCATGCGGATGCATTTTGGCTGAAATGGTCTCCGGGAAGCCTTTGTTCCCAGGCAGAGACTATCATCATCAATTATGGCTAATTCTAGAAGTCTTGGGAACTCCATCTTTCGAAGACTTTAATCAGATCAAATCCAAGAGGGCTAAAGAGTATATAGCAAACTTACCTATGAGGCCACCCTTGCCATGGGAGACCGTCTGGTCAAAGACCGATCTGAATCCAGATATGATAGATTTACTAGACAAAATGCTTCAATTCAATCCTGACAAAAGAATAAGCGCAGCAGAAGCTTTAAGACACCCTTACCTGGCAATGTACCACGATCCTTCAGACGAGCCAGCTGCAGCACTACATCAACTGACTACTGACTACTGACTGCCACCTCGAGGAGACG

5' end common primer:

skpp-1-F ATATAGATGCCGTCCTAGCG(SEQ ID NO.2825)

plate library sorting primers:

skpp-10-R ATATATGGTCACACGCCTGT(SEQ ID NO.2822)

overlapping element sublibrary sorting primers:

skpp-10-1002-R ACACGCCTGTCCTTCGGTTG(SEQ ID NO.803)

assembling a primer:

skpp-102-F TTTGCTTCAGTCAGATTCGC(SEQ ID NO.2830)

skpp-102-R GTTCAATCACTGAATCCCGG(SEQ ID NO.2831)

example 11

Name of DNA Module: BBK602

The experimental scheme is as follows: b + c

Length of DNA module: 693bp

DNA module sequence:

GCGATGTAAATCCTGTGTTTCTTTTATGTATATTTTTTATCAATTTCAATTTAGTTTAGTTTAGTTTCTTTAAAAATAAAATTGTAATTTCTCACATTCTGCCTTCGGGTCTCAAGGGCTGGTTGGTAGACGAATGCGCTTGTTTCTAGCTGAGCGGCAGATGCATGAATTCAATGCAAGAAAATGAGAAAACTTCCAATATTAAAATGTGATTGTTAATTATTAGTATATTTATGACAATATATATATGTACATAGGGTGATTATGAAGATGAGTAAGATGCGAATGGATATGAGGATATGAATCTTTTTAGAGACAATGAAGATGCAAAGAAGTAGTTAAGCACATTGACCTCAGGGATAACTTCGTATAATGTACATTATACGAAGTTATTTGTTATATGCTACTTTTAGTCCGTTGGATTTCTGAAGCAGTATTGGCCACAAAAATTTCTTCATTATTTCTACGATTAGAGTTTCTTCTGGATTTTTCTCGTCCCTGAGACTTACTCCTTTTCAAGTCATTTTCAGCAGCAGCTTTGATTAAATCATATCTTGTGATTTTACTTGGAGAGCTATTGTGATGGGAACTTTGTCTGCAGGTTACCGTATTAGTCAGAGATGTTATGGGCTTAACCATAAGGGAAGAAACTATGGCACGCATGAATGAATTACTGCTTTTACTGCTCATCGCTACTGACTACTGACTGCCACCTCGAGGAGACG

plate library sorting primers:

skpp-6-F CATGTTTAGGAACGCTACCG(SEQ ID NO.2823)

skpp-6-R AATAATCTCCGTTCCCTCCC(SEQ ID NO.2824)

overlapping element sublibrary sorting primers:

skpp-6-602-IN-F AACGCTACCGGGATGCAGTG(SEQ ID NO.2813)

skpp-6-602-R GTTCCCTCCCATCCGGAGAC(SEQ ID NO.403)

assembling a primer:

skpp-102-F TTTGCTTCAGTCAGATTCGC(SEQ ID NO.2830)

skpp-102-R GTTCAATCACTGAATCCCGG(SEQ ID NO.2831)

example 12

Name of DNA Module: BBK603

The experimental scheme is as follows: b + c

Length of DNA module: 688bp

DNA module sequence:

GCGATGGAATGAATTACTGCTTTTACTGCTGTCATCTGTTGAGTTTGCTGAGTCTTCCGTTCTTGTAGTCTTGGCTGGTGTGAGTAACATTACGTATTGTTTCTGTAGAGAAGATAACTTATAACTTCCGTCTTGTCACTAGTTGTTTTAATTATATGTTTGTTACAAGTTTTTATGTATAGATATATGTTACAACTTTTAGAAGTATTGTGGACATAACAGAGAGAAGGGAATGTTGCTATATTTATATATTTTATATATTGCCCTCAAAACTGTATCTCATCTTTTTTTCTGCAATCCTTGCTTTCGTAATCTAAGTGTGGGTGACATGCTAAATCCGCGGCTTTCCTTAAGCTTTTTCACAAACCATCTATTATGTCCAGAAGCTCCGCTGATTATCCGTGGAAAAGGGTCTTATTCTGCCGTAGGAAACTTCTTCTCGAGAAAAAAGTGAGTTATAACTTCGTATAATGTACATTATACGAAGTTATCACCCGCGGCGCTCTCGTAAAACCCGAGGGCCGATTCTTTCCCTCCGTGGAACAATCGGCCCCGCGGCGTCAGAGGGTATTATCTCCGCAAGTGATAGAACTCACATTTATTCTACGTTCTGCATGGTTTTGATTATCTTCTTTTGCACGATGTCACATACTTTTTCGATTACCGTCCATGGCCCCCCCCCCATCGC

plate library sorting primers:

skpp-6-F CATGTTTAGGAACGCTACCG(SEQ ID NO.2823)

skpp-6-R AATAATCTCCGTTCCCTCCC(SEQ ID NO.2824)

overlapping element sublibrary sorting primers:

skpp-6-603-IN-F AACGCTACCGTAATGCAGTG(SEQ ID NO.2814)

skpp-6-603-R GTTCCCTCCCACCTTCAGGT(SEQ ID NO.404)

assembling a primer:

skpp-103-F GTCGAGTCCTATGTAACCGT(SEQ ID NO.2832)

skpp-103-R CAGGGGTCGTCATATCTTCA(SEQ ID NO.2833)

as a result: gel electrophoresis of the assembled products of examples 1-12 is shown in FIG. 9. The lanes in FIG. 9 are DL2000marker, 50bp ladder marker (ladder marker), BBK1002(A + C), BBK1001(A + C), BBK903(C), BBK902(C), BBK803(A), BBK802(A), BBK702(B), BBK701(B), BBK603(B + C), BBK602(B + C), 50bp ladder marker, BBK516 (original protocol), BBK515 (original protocol), respectively, from left to right. The DNA components except BBK602 were assembled successfully.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Hua Dagene institute

<120> oligonucleotide and use thereof

<130>PIOC147487PCN

<160>47

<170>PatentIn version 3.3

<210>2801

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2801

atagtgctgt agtggaaccg 20

<210>2802

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2802

ttcttactag agactgcgcc 20

<210>2803

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2803

cgtgtagtgt gaatatgcgg 20

<210>2804

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>4

gagtcaatga ttgagcctgc 20

<210>2805

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2805

gaaccattcc tttgctgacc 20

<210>2806

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2806

actgtgttag cctcgtttca 20

<210>2807

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2807

gattgaaacg gtgagcagtg 20

<210>2808

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2808

cgtctatgtt tgatcgacgc 20

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2809

gttccgttaa ttcggcagtg 20

<210>2810

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2810

tcagaagaag cgggagaatg 20

<210>2811

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2811

gatatagagc ctccgattcg 20

<210>2812

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2812

gatatagagc gttttggtcg 20

<210>2813

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2813

aacgctaccg ggatgcagtg 20

<210>2814

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2814

aacgctaccg taatgcagtg 20

<210>2815

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2815

tccgacgggg agtatatact 20

<210>2816

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2816

tactaactgc ttcaggccaa 20

<210>2817

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2817

atgatcctat gcgtctgtgt 20

<210>2818

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2818

gtacatgaaa cgatggacgg 20

<210>2819

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2819

ctggtatagt ctcctcagcg 20

<210>2820

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2820

ggcgagagga gatatagagc 20

<210>2821

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2821

aatatcgaac agctgtgcac 20

<210>2822

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2822

atatatggtc acacgcctgt 20

<210>2823

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2823

catgtttagg aacgctaccg 20

<210>2824

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2824

aataatctcc gttccctccc 20

<210>2825

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2825

atatagatgc cgtcctagcg 20

<210>2826

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2826

gagccatgtg aaatgtgtgt 20

<210>2827

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2827

cggactaaag gatcgagtca 20

<210>2828

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2828

cgtatacgta agggttccga 20

<210>2829

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2829

catcggataa cacaaagcgt 20

<210>2830

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2830

tttgcttcag tcagattcgc 20

<210>2831

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2831

gttcaatcac tgaatcccgg 20

<210>2832

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2832

gtcgagtcct atgtaaccgt 20

<210>2833

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2833

caggggtcgt catatcttca 20

<210>2834

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2834

gcttattcgt gccgtgttat 20

<210>2835

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>2835

tacttttgat tgctgtgccc 20

<210>2836

<211>581

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2836

ggtctcctcg agtacagcta tcttggttca gacgtgagaa gtgatgcaca gcagctgtat 60

cctttattta ttggagcttc cgaaacttcc caagaaaagt gaagaaaaaa ccaggaaaat 120

caaaagacat gacgcatata atgtacacct tttttttttc gcggccaaaa atttaaccta 180

ctcatctaga tcagtggttg gtagagcagt attccgtcat ttttcatgtc caggttcgaa 240

gctgacctgg tattatccgg ttttttttct tgttgaccaa taggaaaaaa aaaaattaaa 300

ataaagtcac agaaattcaa tgaatattat aaatttttct tgtttgtttc cctagttgtt 360

atttttataa aaaaaattct tgtagacaat aaaataagaa atgcccattt tgtaacttag 420

cgaaagatgc ccagtacatc ccttttacac ccgtgcatta aaggtgtttg ggtttaatag 480

gagctttatc atatctcttt gatttttttt ctgctgtcct cggcttgagg gactcacaga 540

gatctggaaa ttttcagatt gtcagtgctt aggatgagac c 581

<210>2837

<211>578

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2837

ggtctctagg atgggttgtc agtagacggt ggccgccgtg gatgggaaat ctcatacgtt 60

tacacacata gtgtttggaa attaatagta gcaatagcta tctggctact gttttaaagt 120

attagcccgt tctcagtgct tcttttttaa ggaataacaa cggcaagacc aaagatatat 180

caaatatggc taagcaatct ctaggtatgt ttggaggata cgaataacga tagaaaacat 240

gagtgaattt ccgtccacga aaaaatgtta acataaaatg caagagaaca attaatcgaa 300

taatgttaaa ttattgtaaa acaatgtgta tgatgaggag gaatgtacct aagccaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaagaaa cagcttttgc atattcaatc caggcatagg 420

gcgactattt agcactcaac gatttttaag cttgtgtatt gctgacataa attccggctt 480

tagaatccaa tattgaaaaa cgtgagtacg cagaggagat agcgcgccag cactacatca 540

actgactact gactactgac tgccacctcg aggagacc 578

<210>2838

<211>579

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2838

ggtctcaaga aggcgttctt ccagcataat ttcacatgtg gaaccggaga cttttgaaga 60

tgaaaatgac cagcaacttc taccaaatat gaatgctact tgggtagacc aacgcggcgc 120

ttggattatt catgtggtca ttatcatact gctgaaacta ttttataatt tatttcctgg 180

cgtcactacc gaatggagct ggaccttaac taatatgaca tatgttattg ggtcctatgt 240

catgttccat ctgattaagg gtaccccttt cgatttcaat ggtggtgctt atgacaactt 300

gacgatgtgg gaacaaattg acgacgagac tttatatact ccttcaagaa aatttttgat 360

tagtgtcccg atcgccctat tcttagttag tactcattat gctcactatg atttgaaatt 420

gttttcatgg aattgttttt tgacaacctt tggtgctgtt gtcccaaagt tacctgttac 480

tcatagatta aggatttcta tccctggcat taccggcaga gctcaaatct catgaacaat 540

aacttcgtat aatgtacatt atacgaagtt attgagacc 579

<210>2839

<211>595

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2839

ggtctcgtta ttagtttatt tcaatcttgt atctagatgt ttttgctata tttatatttt 60

ctgggccaaa tcactaacgc tttgaaattt caaaagccaa caacggattt cggacttcat 120

ttatgtaaat gaaaaagaat gtgaacaaaa attgaaaaaa ttccatatta atgcaccttc 180

taaacctatt tgttgaaatt gtatttaatt catataatca tattctattc aaaaagaata 240

acagatagta ttagcactat ttcacgtact gcttctaata ctattatcac aaaatagaca 300

tcggaaaact caaaaataaa taacttcgta taatgtacat tatacgaagt tatataactt 360

cgtataatgt acattatacg aagttatgta tttatgtttt gtcattcttt tctacataat 420

cttgaaacta ggtagatcta caattgaaaa gtaaatacta acattattta ctaaatttaa 480

gttagaaatc ggcacgaaaa aaatttgaca gattacgaga gtccagccaa aatatgagta 540

tattactatt tccccttggt gaaagaaatg aaagatgtta ttttttaccg agacc 595

<210>2840

<211>632

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2840

ggtctcggat ccttcttttg cacgatgtca catacttttt cgattaccgt cgctttacac 60

agtaactgtt taaaacgagc gttcttaatt aataactgta ggagagcagg ctttcggctt 120

tctttatgag aaataaggac tactccagct tgccaagttt tatccagtta cacaaacgct 180

caagatcgcc tcgctaaaatagatgaaaaa ataagaaaaa tgaaatgttc ataacttcgt 240

ataatgtaca ttatacgaag ttataactta atcccttttg gatttatcat tgaaagatcg 300

ctgatttatt cttttccaat acgcctcgta attctctttc aatgtcaaag gcgccagatt 360

gtaagaatct gtcatgtaaa ataatctgac aatacgtagt tttaaaaacc aattgattaa 420

accaagctct cttatgatgc caactataaa gaagtacaag ctaccggtta aagcaccaac 480

gctgaatccc acgattactt gatctaaatt gtggtagtgc aagtaaactc tggaaaagca 540

aacgcaaaac gataataaag ccaatgcacc agaaaaaatg catttttcta agaagtttag 600

attcttccag gaagtgtata tcttcagaga cc 632

<210>2841

<211>631

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2841

ggtctctctt cagagagtta taggtaaaac aaaaccccat gaattgggag tgtgcactgg 60

gcatcccgta accggatctt atagtgtcat tttggaacga cgcaccgaac gatacggggc 120

gtggctgttt tattatattc ttgatcacgt tattgaatat ttcgttcatc aattggccaa 180

aagcaacgat acaagcttcc aactcacggg tgatgataaa ccacgacaaa taaaaagcta 240

gcactaggat gggcatcagc gagaaatatg cactaaggaa tgatagaaag tcatgcgaat 300

catagagaat gtatgtgtca tcgaatggta taacatttgg atttggattt attgcagcgg 360

cggtactatt catgatatga tccgattcaa aacaaatttt gatgtagata aaatgcctgg 420

tataatgagt ggtagagaag aaaattagac gaaaagaatg agaacgaaga aatgatttaa 480

agtgatcaca gtggcaatca ctttcccctt tttaacgcgt gttgagcaat cgattgatgt 540

ctctaccata tgtagcattt tctcagaagg caaacttttc tttttctttc tacttttcca 600

acaagaaatt ttaagttttc ccagagagac c 631

<210>2842

<211>523

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2842

ggtctcaatc aaagaggcga tatcaacacc ttttatccag cactattcaa cagtgaatgg 60

gctcccaagt aagtcttggc attgtgcttt ctattcttaa gtattaagta gaagttttgt 120

ttactgggtt tgtttattcc tggctagatg ttcgcattcg ttttctagtt gaccatattt 180

accaaatatt cacaactaat acccagccaa ggtagtctaa aagctaattt ctctaaaagg 240

gagaaagttg gtgatttttt atctcgcatt attatatatg caagaatagt taaggtatag 300

ttataaagtt ttatcttaat tgccacatac gtacattgac acgtagaagg actccattat 360

ttttttcatt ctagcatact attattcctt gtaacgtccc agagtattcc atttaattgt 420

cctccatttc ttaacggtga cgaaggatca ccatacaaca actactaaag attatagtac 480

actctcacct tgcaactatt tatctgacat ttgccttgag acc 523

<210>2843

<211>526

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2843

ggtctctgcc ttacttttat ctccagcttc ccctcgattt tatttttcaa tttgatttct 60

aaagcttttt gcttaggcat accaaaccat ccactcattt aacaccttat tttttttttc 120

gaagacagca tccaacttta tacgttcact accttttttt ttacaacaat ttcattcttc 180

atcctatgaa atgacgaaaa taaccagaga tgtttcgata acgacagaaa attcaaaatc 240

cacatctgga tcggcaacgg cctcttcggc ctctttacct gagaacgacc acccaatatt 300

ccatcaacct agagctcgta tccgtagcgg cagcttattc atcgaaggat ctgattcatt 360

tccatcatca gaagtgaagt catacaatgt ttatattgat gatagcaagt atagtgaaat 420

cctcaaagga gatacaaatt caagtagtac tgatggtaaa caagtctttg aagatgctag 480

agatgacaat ttccatcagg aatcacatag agatctagag gagacc 526

<210>2844

<211>567

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2844

cgtctcctcg agtacagcta tcttggttca gacgtgagaa gtgatgcaca ggtcgacttt 60

tcttcacttt tctttcaaca attcaaagat ggctagaacc ataacttttg atatcccttc 120

ccaatataaa ctcgtagatt taataggtga gggagcgtac ggaacagtat gttcagcaat 180

tcataagcct tccggcataa aggtagctat caagaaaata caaccgttta gcaaaaaatt 240

gtttgttaca agaactatac gtgagatcaa gcttttacgg tatttccatg aacacgaaaa 300

cataataagt atattggata aagtaaggcc agtatccata gacaaactaa acgctgttta 360

tttagtcgaa gagttgatgg aaaccgattt acaaaaagta attaataatc agaatagcgg 420

gttttccact ttaagtgatg accatgttca atactttaca taccaaatcc tcagagcctt 480

aaagtctatt cacagtgcac aagttatcca tagagacata aagccatcaa acctgttact 540

aaattccaat tgtgatctca agagacg 567

<210>2845

<211>563

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2845

cgtctctctc aaagtctgcg attttggact agctagatgc ttggcctcat catcagacag 60

cagagaaaca ttggtaggat tcatgacgga gtacgtcgca acgcgatggt acagggcacc 120

cgagataatg ctaacttttc aagagtacac aactgcgatg gatatatggt catgcggatg 180

cattttggct gaaatggtct ccgggaagcc tttgttccca ggcagagact atcatcatca 240

attatggcta attctagaag tcttgggaac tccatctttc gaagacttta atcagatcaa 300

atccaagagg gctaaagagt atatagcaaa cttacctatg aggccaccct tgccatggga 360

gaccgtctgg tcaaagaccg atctgaatcc agatatgata gatttactag acaaaatgct 420

tcaattcaat cctgacaaaa gaataagcgc agcagaagct ttaagacacc cttacctggc 480

aatgtaccac gatccttcag acgagccagc tgcagcacta catcaactga ctactgacta 540

ctgactgcca cctcgaggag acg 563

<210>2846

<211>725

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2846

gcgatgtaaa tcctgtgttt cttttatgta tattttttat caatttcaat ttagtttagt 60

ttagtttctt taaaaataaa attgtaattt ctcacattct gccttcgggt ctcaagggct 120

ggttggtaga cgaatgcgct tgtttctagc tgagcggcag atgcatgaat tcaatgcaag 180

aaaatgagaa aacttccaat attaaaatgt gattgttaat tattagtata tttatgacaa 240

tatatatatg tacatagggt gattatgaag atgagtaaga tgcgaatgga tatgaggata 300

tgaatctttt tagagacaat gaagatgcaa agaagtagtt aagcacattg acctcaggga 360

taacttcgta taatgtacat tatacgaagt tatttgttat atgctacttt tagtccgttg 420

gatttctgaa gcagtattgg ccacaaaaat ttcttcatta tttctacgat tagagtttct 480

tctggatttt tctcgtccct gagacttact ccttttcaag tcattttcag cagcagcttt 540

gattaaatca tatcttgtga ttttacttgg agagctattg tgatgggaac tttgtctgca 600

ggttaccgta ttagtcagag atgttatggg cttaaccata agggaagaaa ctatggcacg 660

catgaatgaa ttactgcttt tactgctcat cgctactgac tactgactgc cacctcgagg 720

agacg 725

<210>2847

<211>688

<212>DNA

<213> Artificial sequence

<220>

<223> DNA Module sequences

<400>2847

gcgatggaat gaattactgc ttttactgct gtcatctgtt gagtttgctg agtcttccgt 60

tcttgtagtc ttggctggtg tgagtaacat tacgtattgt ttctgtagag aagataactt 120

ataacttccg tcttgtcact agttgtttta attatatgtt tgttacaagt ttttatgtat 180

agatatatgt tacaactttt agaagtattg tggacataac agagagaagg gaatgttgct 240

atatttatat attttatata ttgccctcaa aactgtatct catctttttt tctgcaatcc 300

ttgctttcgt aatctaagtg tgggtgacat gctaaatccg cggctttcct taagcttttt 360

cacaaaccat ctattatgtc cagaagctcc gctgattatc cgtggaaaag ggtcttattc 420

tgccgtagga aacttcttct cgagaaaaaa gtgagttata acttcgtata atgtacatta 480

tacgaagtta tcacccgcgg cgctctcgta aaacccgagg gccgattctt tccctccgtg 540

gaacaatcgg ccccgcggcg tcagagggta ttatctccgc aagtgataga actcacattt 600

attctacgtt ctgcatggtt ttgattatct tcttttgcac gatgtcacat actttttcga 660

ttaccgtcca tggccccccc cccatcgc 688

Claims

1. An oligonucleotide, comprising:

a gene coding region;

a first flanking sequence formed 5' of the coding region of the gene; and

a second flanking sequence formed 3' of the coding region of the gene,

wherein a plate bank sorting primer binding sequence, a component sub-bank sorting primer binding sequence and an enzyme cutting site are sequentially formed on the first flanking sequence from a 5 'end to a 3' end, and the enzyme cutting site, the component sub-bank sorting primer binding sequence and the plate bank sorting primer binding sequence are sequentially formed on the second flanking sequence from the 5 'end to the 3' end, wherein the plate bank sorting primer binding sequence, the component sub-bank sorting primer binding sequence and the enzyme cutting site in the first flanking sequence satisfy the following conditions:

(b) at least one part of the enzyme cutting sites are formed inside the sorting primer binding sequences of the component sub-library, and the enzyme cutting sites are connected with the gene coding regions;

the plate subpool sorting primer binding sequence, the assembly subpool sorting primer binding sequence and the enzyme cutting site in the second flanking sequence satisfy the following conditions:

(c) the plate sub-library sorting primer binding sequence shares a partial overlap sequence with the component sub-library sorting primer binding sequence.

2. The oligonucleotide of claim 1, wherein the oligonucleotide is 100 to 300nt in length.

3. The oligonucleotide of claim 1, wherein the cleavage site is 6 to 12nt in length.

4. The oligonucleotide of claim 1, wherein the cleavage site is a type II restriction enzyme cleavage site.

5. The oligonucleotide of claim 1, wherein the common primer binding sequence, the modular subsubstrate selector primer binding sequence, and the plate subsubstrate selector primer binding sequence are 15-25nt in length.

6. The oligonucleotide of claim 1, wherein the common primer binding sequence is the complement of SEQ id No. 2825.

7. The oligonucleotide of claim 1, wherein the plate library sorting primer binding sequence is a complementary sequence of at least one of SEQ ID No.2815 to SEQ ID No. 2824.

8. The oligonucleotide of claim 1, wherein the modular sublibrary sorting primer binding sequence is a complementary sequence of at least one of SEQ ID No.1 to SEQ ID No. 2814.

9. The oligonucleotide according to claim 1, wherein the length of the overlapping sequence is no greater than 10 nt.

10. Use of the oligonucleotide of any one of claims 1 to 9 in DNA chip synthesis.

11. A method of synthesizing DNA, comprising:

synthesizing an oligonucleotide library by using the chip;

assembling based on the oligonucleotide library so as to obtain a target DNA,

wherein the library of oligonucleotides comprises a plurality of oligonucleotide strands, said oligonucleotide strands being the oligonucleotides of any one of claims 1-9.