CN102409045B - Tag library constructing method based on DNA (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter - Google Patents
Tag library constructing method based on DNA (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter Download PDFInfo
- Publication number
- CN102409045B CN102409045B CN 201010299257 CN201010299257A CN102409045B CN 102409045 B CN102409045 B CN 102409045B CN 201010299257 CN201010299257 CN 201010299257 CN 201010299257 A CN201010299257 A CN 201010299257A CN 102409045 B CN102409045 B CN 102409045B
- Authority
- CN
- China
- Prior art keywords
- dna
- label
- index
- joint
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
Abstract
According to the invention, based on a DNA (deoxyribonucleic acid) tag library preparation method provided by a solexa sequencing platform of the illumine corporation, a special tag sequence with length of 6 bp is designed, the tags are embedded into DNA adapters, and the DNA adapters are connected to introduce the tag sequence, thus a library construction method of a DNA tag library is successfully constructed, and the constructed DNA tag library can be applied to solexa DNA sequencing.
Description
Technical field
The present invention relates to the nucleic acid sequencing technical field, particularly DNA tag library preparation method.In addition, the invention still further relates to label technique, and realize that a plurality of samples make up the method for tag library in same reaction system.Method of the present invention is specially adapted to s-generation sequencing technologies, especially the solexa sequencing technologies.
Background technology
The Solexa dna sequencing platform that Illumina company provides, can in a reaction, add simultaneously four kinds and be with fluorescently-labeled Nucleotide, adopt order-checking (the Sequencing By Synthesis while synthesizing, SBS), has required sample size few, high-throughput, high precision has automatization platform simple to operation and the characteristics [1-4] such as powerful.Library construction at first needs the purpose fragment is carried out end reparation, 3 ' terminal " A " base that connects in the purpose fragment, 3 ' terminal purpose fragment with the A base is connected with DNA joint (being also referred to as adapter), by the PCR reaction purpose fragment is increased, reclaim at last the purpose fragment library of containing the DNA joint, see Fig. 1.Purpose fragment library and the DNA joint above the sequence testing chip are hybridized, after increasing by bridge-type PCR, and synthetic limit order-checking backmost.In each working cycle, fluorescently-labeled nucleosides and polysaccharase are added in the single molecule array.Complementary nucleosides and first base pairing of nucleotide fragment join on the primer by enzyme.Unnecessary nucleosides is removed.Each single strand dna is extended by the pairing of complementary base like this, and for the laser excitation of the specific wavelength of the every kind of base mark in conjunction with upper nucleosides, this mark can discharge fluorescence, and the fluorescent signal of collecting is at last translated into base sequence.Present this DNA banking process can apply to various research fields according to demand, such as genomic De Novo order-checking, genome resurvey order, transcribe group order-checking and the order-checking of apparent gene group etc.
Also released DNA label (being also referred to as index) banking process based on above-mentioned banking process illumina company, as shown in Figure 2.Build in the flow process of storehouse at the DNA label, the PCR process has been used 3 PCR Tag primers, imports label by PCR and comes constructed dna tag library [5].Can be with the method for sample mixing order-checking thereby disclose a kind of source of sequence label labeling nucleic acid sample of using among patent application WO2005068656A1 and the WO2008093098A2, can specific nucleotide sequence (sequence label) be imported in the library by PCR by the process of PCR, PCR Tag primer sequence sees Table 1.These libraries with label can be mixed arbitrarily according to demand, then check order by solexa order-checking instrument, at last data are classified by sequence label.
Label (index) sequence that table 1 illumina company provides and corresponding PCR Tag primer (indexN PCR primer) sequence
But the method for the tag library that illumina company provides preparation exists some defectives: the first, at present illumina company 12 length only are provided is the sequence label of 6bp, the comparatively small amt of label, along with the increase of solexa sequencing throughput, can not mix order-checking to great amount of samples will be a huge defective; The second, at present the label banking process that provides of illumina company is by the PCR reaction sequence label to be imported in the purpose fragment library, need 3 PCR primer pair purpose fragments increase (two public primer and a strip label PCR primer, such as table 1), and pcr amplification efficient is not high.Three, the label banking process center tap that provides of illumina company does not comprise sequence label, each tag library need to react to import sequence label by a PCR, then needing to cut glue for each tag library reclaims, then mix in the label purpose fragment library that will cut after glue reclaims, so not only waste time and energy, and expense is also higher.
Therefore, sequence and label introducing method to label are optimized and improve, the efficient that label is introduced improves, enlarge the quantity of sequence label, could satisfy high-throughout requirement of building the storehouse, the present situation that improves constantly to adapt to sequencing throughput takes full advantage of the production capacity of order-checking instrument, reduces the cost of order-checking.
Summary of the invention
The present invention comes the constructed dna tag library with in the label intercalation of DNA joint by connecting DNA label joint, and the product that connects upper DNA label joint is mixed, and a PCR reaction just can be finished the structure of all tag libraries that needs are mixed.Can not only improve the sequencing throughput of present DNA sample, also can improve the efficient of library preparation and the recognition rate of label, greatly reduce the order-checking expense in single library.
The DNA tag library preparation method's who provides in view of the solexa order-checking platform of present illumina company deficiency, the present invention has improved the preparation method of DNA tag library, the joint that has designed length and be the sequence label of 6bp uniqueness and contained described sequence label, the connection of the DNA joint by comprising sequence label imports sequence label, successful foundation the banking process of DNA tag library, and be applied to the solexa dna sequencing, improved the efficient of the preparation of DNA tag library, increase the sequencing throughput of DNA sample, reduced the solexa order-checking expense of single sample.
In the present invention, label design at first needs to consider identifiability between the sequence label and the problem of recognition rate, then need to consider that sequence label mixes the GT in each site afterwards and the equilibrium problem of AC base contents, consider at last repeatability and the accuracy of data output.In the process of tag design, the present invention fully takes into account above several factor, has avoided simultaneously the nucleotide sequence of label base consecutive identical more than 3 or 3 to occur, can reduce like this sequence in building-up process or the error rate in the order-checking process.Avoid simultaneously DNA label joint self to form hairpin structure as far as possible, improve the joint efficiency of DNA label joint.
The present invention is optimized the DNA joint sequence that illumina provides, and introduces sequence label in joint, and the connection by DNA label joint imports sequence label in the purpose library.In the PCR reaction after joint connects, also just need not to use extra label PCR primer, thereby saved the synthetic step of primer, reduced the difficulty of PCR reaction, improved the specificity of PCR reaction.So far, import banking process and the sequence label of label by these DNA label joints, not relevant report.DNA label joint after the optimization is compared with the DNA joint of illumina company, has improved the efficient that joint connects, and has improved the quantity of recognition efficiency and the label of sequence label.The DNA label that is illustrated in figure 3 as illumina company is built the storehouse schema, the DNA tag library construction process experiment flow figure of Fig. 4 for connecting based on joint after optimizing.
The present invention is based on the Solexa Paired End order-checking platform that present illumina company provides, designing a segment length is the specific label nucleotide sequence of 6bp.By the amplification efficiency of test dna label PCR primer and the recognition rate of label nucleotide sequence, optimize at last and DNA sequence label (such as table 2, the DNA sequence label DNA Index-N of 6bp) and DNA label joint sequence (DNA Index-NF/R_adapter) that to filter out 67 length be 6bp.These length are that the difference between the label of 6bp has 3 bases at least, i.e. at least 3 base sequence differences.Order-checking mistake or resultant fault appear in any one base in 6 bases of label, do not have influence on the final identification of label.
Table 2 length is the DNA sequence label (DNA Index-N) of 6bp and forward and the reverse sequence (DNA Index-NF_adapter and DNA Index-NR_adapter) of corresponding DNA label joint thereof, and the direction of sequence shown in the table all is 5 '-3 ' directions.
Use the PrimerSelect software of Lasergene, judge affinity parameters between the duplex by analyzing the Energy value that forms between the two sequences, the result of the larger expression duplex of absolute value of Energy value (kcal/mol) is more stable, below be respectively the Energy value of the avidity of having analyzed above-mentioned DNA label joint, illustrate that the structure of these DNA label joints formation is highly stable.
Secondary structure and Energy value that the DNA label joint of prediction forms.The most stable joint sequence (the most stable dimer overall).
DNA Index-1 joint
The most stable dimer overall：19bp，-35.5kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCAAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTGGTT 5′
DNA Index-2 joint
The most stable dimer overall：19bp，-34.0kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAACTTGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTGAAC 5′
DNA Index-3 joint
The most stable dimer overall：19bp，-33.4kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGAGTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTCTCA 5′
DNA Index-4 joint
The most stable dimer overall：19bp，-33.0kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAATAACT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTATTG 5′
DNA Index-5 joint
The most stable dimer overall：19bp，-36.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTACACGCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATGTGCG 5′
DNA Index-6 joint
The most stable dimer overall：19bp，-34.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTACCTCTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATGGAGA 5′
DNA Index-7 joint
The most stable dimer ove rall：19bp，-36.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTACGGAAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATGCCTT 5′
DNA Index-8 joint
The most stable dimer overall：19bp，-35.2kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGAAGGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCTTCC 5′
DNA Index-9 joint
The most stable dimer overall：19bp，-38.1kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCCGTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCGGCA 5′
DNA Index-10 joint
The most stable dimer overall：19bp，-36.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCGAGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCGCTC 5′
DNA Index-11 joint
The most stable dimer overall：19bp，-36.7kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGCTGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCCGAC 5′
DNA Index-12 joint
The most stable dimer overall：19bp，-36.4kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTGCCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCACGG 5′
DNA Index-13 joint
The most stable dimer overall：19bp，-32.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTATAATCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATATTAG 5′
DNA Index-14 joint
The most stable dimer overall：19bp，-33.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTATGTCAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATACAGT 5′
DNA Index-15 joint
The most stable dimer overall：19bp，-35.1kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTATTCCTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATAAGGA 5′
DNA Index-16 joint
The most stable dimer overall：19bp，-34.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCAACACT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTTGTG 5′
DNA Index-17 joint
The most stable dimer overall：19bp，-34.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCACAAGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTGTTC 5′
DNA Index-18 joint
The most stable dimer overall：19bp，-37.2kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCACGGTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTGCCA 5′
DNA Index-19 joint
The most stable dimer overall：19bp，-34.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCACTCAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTGAGT 5′
DNA Index-20 joint
The most stable dimer overall：19bp，-37.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAACGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGTTGC 5′
DNA Index-21 joint
The most stable dimer overall：19bp，-37.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAGGAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGTCCT 5′
DNA Index-22 joint
The most stable dimer overall：19bp，-40.5kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGCCTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGCGGA 5′
DNA Index-23 joint
The most stable dimer overall：19bp，-34.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTTACT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGAATG 5′
DNA Index-24 joint
The most stable dimer overall：19bp，-35.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAATAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCTTAT 5′
DNA Index-25 joint
The most stable dimer overall：19bp，-39.2kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGCGTCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCGCAG 5′
DNA Index-26 joint
The most stable dimer overall：19bp，-36.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGGTAAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCCATT 5′
DNA Index-27 joint
The most stable dimer overall：19bp，-39.2kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTCGGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCAGCC 5′
DNA Index-28 joint
The most stable dimer overall：19bp，-34.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTAGCTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGATCGA 5′
DNA Index-29 joint
The most stable dimer overall：19bp，-37.5kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTCCGAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGAGGCT 5′
DNA Index-30 joint
The most stable dimer overall：19bp，-34.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGTTGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGACAAC 5′
DNA Index-31 joint
The most stable dimer overall：19bp，-33.4kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTAGTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGAATCA 5′
DNA Index-32 joint
The most stable dimer overall：19bp，-35.5kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGACCTCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTGGAG 5′
DNA Index-33 joint
The most stable dimer overall：19bp，-37.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGAGCCAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTCGGT 5′
DNA Index-34 joint
The most stable dimer overall：19bp，-33.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGAGTATT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTCATA 5′
DNA Index-35 joint
The most stable dimer overall：19bp，-36.0kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGATGGAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTACCT 5′
DNA Index-36 joint
The most stable dimer overall：19bp，-35.4kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGCATAGT 3′
::: :|||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACGTATC 5′
DNA Index-37 joint
The moststable dimer overall：19bp，-42.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCGGCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACGGCCG 5′
DNA Index-38 joint
The most stable dimer overa1l：19bp，-37.2kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGCGTTAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACGCAAT 5′
DNA Index-39 joint
The most stable dimer overall：19bp，-36.1kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAGAAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCTCTT 5′
DNA Index-40 joint
The most stable dimer overall：19bp，-37.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCATGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCGTAC 5′
DNA Index-41 joint
The most stable dimer overall：19bp，-40.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCGCTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCGCGA 5′
DNA Index-42 joint
The most stable dimer overall：19bp，-36.4kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTAGCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCATCG 5′
DNA Index-43 joint
The most stable dimer overall：19bp，-37.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTTCGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCAAGC 5′
DNA Index-44 joint
The most stable dimer overa1l：19bp，-33.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTACATT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACATGTA 5′
DNA Index-45 joint
The most stable dimer overall：19bp，-34.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTATCCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACATAGG 5′
DNA Index-46 joint
The most stable dimer overall：19bp，-34.0kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCTGTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACAGACA 5′
DNA Index-47 joint
The most stable dimer overall：19bp，-39.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGCGCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACACGCG 5′
DNA Index-48 joint
The most stable dimer overall：19bp，-39.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGCGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACACCGC 5′
DNA Index-49 joint
The most stable dimer overall：19bp，-33.7kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTACAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACAATGT 5′
DNA Index-50 joint
The most stable dimer overall：19bp，-34.3kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTGACT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACAACTG 5′
DNA Index-51 joint
The most stable dimer overall：19bp，-35.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTAATCGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATTAGC 5′
DNA Index-52 joint
The most stable dimer overall：19bp，-35.2kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGGAGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATCCTC 5′
DNA Index-53 joint
The most stable dimer overall：19bp，-35.2kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGTGCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATCACG 5′
DNA Index-54 joint
The most stable dimer overall：19bp，-35.4kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTATGCTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATACGA 5′
DNA Index-55 joint
The most stable dimer overall：19bp，-34.5kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAGATT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGTCTA 5′
DNA Index-56 joint
The most stable dimer overall：19bp，-34.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATTCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGTAAG 5′
DNA Index-57 joint
The most stable dimer overall：19bp，-37.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCAGGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGGTCC 5′
DNA Index-58 joint
The most stable dimer overall：19bp，-39.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCGCAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGGCGT 5′
DNA Index-59 joint
The most stable dimer overall：19bp，-34.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTACCT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGATGG 5′
DNA Index-60 joint
The most stable dimer overall：19bp，-36.0kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTCGTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGAGCA 5′
DNA Index-61 joint
The most stable dimer overall：19bp，-35.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCTTAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACGAAT 5′
DNA Index-62 joint
The most stable dimer overall：19bp，-36.1kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGAGAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACCTCT 5′
DNA Index-63 joint
The most stable dimer overall：19bp，-35.8kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCTT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACCAGA 5′
DNA Index-64 joint
The most stable dimer ove rall：19bp，-33.9kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTAATT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACATTA 5′
DNA Index-65 joint
The most stable dimer overall：19bp，-34.0kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTTACTGT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAATGAC 5′
DNA Index-66 joint
The most stable dimer overall：19bp，-33.6kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTTATAAT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAATATT 5′
DNA Index-67 joint
The most stable dimer overall：19bp，-36.1kcal/mol
5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCACT 3′
::: : |||||||||||||||||||
3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAAGGTG 5′
Description of drawings
Fig. 1: the conventional DNA that illumina company provides builds the storehouse schematic flow sheet.
Fig. 2: the conventional DNA label that illumina company provides is built the storehouse schematic flow sheet.
Fig. 3: the DNA label of illumina company is built the storehouse schematic flow sheet.
Fig. 4: the schematic flow sheet of the DNA tag library construction process that connects based on joint after the optimization of the present invention.
Fig. 5: make up 67 DNA tag library electrophoresis result.(a) DNA label joint library test (the electrophoresis detection result (swimming lane 1 and swimming lane 25 are respectively that D2000 makrer and 50bp marker, swimming lane 2 are respectively the library of using DNA label joint index1～index23 structure to swimming lane 24) of index1～index23); (b) ((swimming lane 1 and swimming lane 25 are respectively that D2000 makrer and 50bp marker, swimming lane 2 are respectively the library of using DNA label joint index23～index44 structure to swimming lane 24 to the electrophoresis detection result of index23～index44) in DNA label joint library test, wherein swimming lane 14 does not namely have sample for the negative control of test); (c) DNA label joint library test (the electrophoresis detection result (swimming lane 1 and swimming lane 25 are respectively D2000 makrer and 50bp marker, swimming lane 2 use respectively DNA label joint index45～index67 structure to swimming lane 24 libraries) of index45～index67).
Fig. 6: the result that the DNA tag library of structure uses Agilent2100 to detect.Sample is called Agilent3, and Tu Zhongfeng from left to right represents respectively Marker, the sample fragment size, and Marker, the library clip size of surveying is 284bp, concentration is 32.64ng/ul.Library size and concentration are all qualified.
Embodiment
Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.
One aspect of the present invention provides one group of DNA label, and it is following or be comprised of following that described DNA label comprises: the label of DNA shown in the table 2 differs with it at least 5 in the DNA label of 1 base, or at least 10, or at least 15, or at least 20, at least 25, or at least 30, or at least 35, or at least 40, or 45, or at least 50, or at least 55, or at least 60, or whole 67
Described DNA label preferably comprises the DNA Index-1 of 67 DNA labels shown in the table 2～DNA Index-5 at least, or DNA Index-6～DNA Index-10, or DNA Index-11～DNA Index-15, or DNA Index-16～DNA Index-20, or DNA Index-21～DNA Index-25, or DNA Index-26～DNA Index-30, or DNA Index-31～DNA Index-35, or DNA Index-36～DNA Index-40, or DNA Index-41～DNA Index-45, or DNA Index-46～DNA Index-50, or DNA Index-51～DNA Index-55, or DNA Index-56～DNA Index-60, or DNA Index-61～DNA Index-65, or DNA Index-63～DNA Index-67, perhaps their any two or more combination.
In a specific embodiment of the present invention, in the DNA label provided by the invention, wherein differ replacement, interpolation or deletion that 1 base comprises 1 base in the described label.
The present invention on the other hand, provide described DNA label to be used for the purposes of constructed dna tag library, wherein the DNA label joint of DNA tag library comprises described DNA label at 3 ' end, thereby consist of corresponding separately DNA label joint, described DNA label joint uses as 5 ' joint and the 3 ' joint of DNA tag library simultaneously.
In a specific embodiment of the present invention, provide described DNA label to be used for the purposes of constructed dna tag library, in the 3 ' end in the wherein said DNA label insertion DNA label joint, by or be not connected to 3 ' end of DNA joint by connexon, preferably be not connected to 3 ' end of DNA joint by connexon.
The present invention provides the one group of DNA label joint that contains claim 1 or 2 described DNA labels on the other hand, wherein the DNA label joint of DNA tag library comprises described label at 3 ' end, and simultaneously as 5 ' joint and 3 ' joint, it is following or be comprised of following that described DNA label joint comprises: 67 DNA label joints and its DNA sequence label that comprises shown in the table 2 differ at least 5 in the DNA label joint of 1 base, or at least 10, or at least 15, or at least 20, at least 25, or at least 30, or at least 35, or at least 40, or 45, or at least 50, or at least 55, or at least 60, or whole 67
Described DNA label joint preferably comprises DNA Index-1F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-5F/R_adapter at least, or DNA Index-6F/R_adapte～DNA Index-10F/R_adapter, or DNA Index-11F/R_adapte～DNA Index-15F/R_adapter, or DNA Index-16F/R_adapte～DNA Index-20F/R_adapter, or DNA Index-21F/R_adapte～DNA Index-25F/R_adapter, or DNA Index-26F/R_adapte～DNA Index-30F/R_adapter, or DNA Index-31F/R_adapte～DNA Index-35F/R_adapter, or DNA Index-36F/R_adapte～DNA Index-40F/R_adapter, or DNA Index-41F/R_adapte～DNA Index-45F/R_adapter, or DNA Index-46F/R_adapte～DNA Index-50F/R_adapter, or DNA Index-51F/R_adapte～DNA Index-55F/R_adapter, or DNA Index-56F/R_adapte～DNA Index-60F/R_adapter, or DNA Index-61F/R_adapte～DNA Index-65F/R_adapter, or DNA Index-66F/R_adapte～DNA Index-67F/R_adapter, perhaps their any two or more combination.
In a specific embodiment of the present invention, in the described DNA label joint, describedly differ replacement, interpolation or the deletion that 1 base comprises 1 base in the label.
Of the present invention one further aspect in, the purposes that provides described DNA label joint to be used for the constructed dna tag library, preferably described DNA label joint is simultaneously as 5 ' joint and the 3 ' joint of DNA tag library.
In another aspect of this invention, provide the DNA tag library that makes up by DNA label joint provided by the present invention.
The present invention provides a kind of construction process of tag library on the other hand, and described method is characterised in that with the DNA joint that comprises label and makes up tag library.
In the further aspect of the present invention, provide a kind of construction process of tag library, it comprises:
1) provide n DNA sample, n is the integer of integer and 1≤n≤67, and preferably n is integer and 2≤n≤67, and described DNA sample includes but not limited to people DNA sample from all eucaryons and procaryotic DNA sample;
2) human gene group DNA is interrupted, the method that wherein interrupts includes but not limited to that ultrasonic wave interrupts method, and the DNA band after interrupting is concentrated on about 180bp;
3) the terminal reparation;
4) dna fragmentation 3 ' end adds " A " base;
5) connect DNA label joint, wherein preferably each label joint is connected to the two ends of dna fragmentation;
6) with step 5) the connection product that obtains carries out gel purification, and preferably carry out electrophoresis by 2% agarose gel and reclaim, and the recovery product of each DNA sample is mixed;
7) step 6 is used in PCR reaction) the mixture of recovery product as template, under the condition of purpose nucleic acid that is suitable for increasing, carry out pcr amplification, the PCR product is carried out glue reclaims purifying, preferably reclaim the purpose fragment of 280～300bp.
In a specific embodiment of the present invention, in the invention provides a kind of construction process of tag library, it is following or be comprised of following that described DNA label joint comprises: 67 DNA label joints and its DNA sequence label that comprises shown in the table 2 differ at least 5 in the DNA label joint of 1 base, or at least 10, or at least 15, or at least 20, at least 25, or at least 30, or at least 35, or at least 40, or 45, or at least 50, or at least 55, or at least 60, or whole 67
Described DNA label joint preferably comprises DNA Index-1F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-5F/R_adapter at least, or DNA Index-6F/R_adapte～DNA Index-10F/R_adapter, or DNA Index-11F/R_adapte～DNA Index-15F/R_adapter, or DNA Index-16F/R_adapte～DNA Index-20F/R_adapter, or DNA Index-21F/R_adapte～DNA Index-25F/R_adapter, or DNA Index-26F/R_adapte～DNA Index-30F/R_adapter, or DNA Index-31F/R_adapte～DNA Index-35F/R_adapter, or DNA Index-36F/R_adapte～DNA Index-40F/R_adapter, or DNA Index-41F/R_adapte～DNA Index-45F/R_adapter, or DNA Index-46F/R_adapte～DNA Index-50F/R_adapter, or DNA Index-51F/R_adapte～DNA Index-55F/R_adapter, or DNA Index-56F/R_adapte～DNA Index-60F/R_adapter, or DNA Index-61F/R_adapte～DNA Index-65F/R_adapter, or DNA Index-66F/R_adapte～DNA Index-67F/R_adapter, perhaps their any two or more combination.
In a specific embodiment of the present invention, in the invention provides a kind of construction process of tag library, describedly differ replacement, interpolation or the deletion that 1 base comprises 1 base in the label.
In a specific embodiment of the present invention, in the invention provides a kind of construction process of tag library, step 7 wherein) primer that uses in the PCR reaction comprises
PE PCR Primers 1.0：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT CT; With
PE PCR Primers 2.0：
CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT。
The present invention further on the other hand in, the tag library that provides the construction process by tag library provided by the invention to make up.
Paired End DNA oligonucleotide sequence:
PE PCR Primers 1.0
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
PE PCR Primers 2.0
CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
Main laboratory apparatus and reagent
The structure of embodiment 1DNA tag library
1. method steps
1.1DNA interrupt
Use Covaris to smash instrument people's Whole Blood Genomic DNA 5ug and interrupt (parameter setting: Duty cycle (duty ratio)-20% in 6 minutes; Intensity (intensity)-5.0; Bursts per second (pulse p.s.)-200; Duration (time length)-40seconds; Mode (pattern)-Frequency sweeping (frequency sweeping); Power (power)-33-34W; 6 ℃ of Temperature (temperature)-5.5to), make its main band that in agarose electrophoresis, shows concentrate on [5] about 180bp.
1.2 terminal the reparation
Mix according to following proportioning ready reaction:
Dna fragmentation 35 μ L after interrupting
T4 dna ligase damping fluid 50 μ L
DNTPs mixed solution 4 μ L
T4 archaeal dna polymerase 5 μ L
Klenow archaeal dna polymerase 1 μ L
Cumulative volume 100 μ L
The comfort type constant-temperature mixer is transferred to 20 ℃, and then reaction 30min carries out purifying with QIAquick PCR purification kit, at last sample is dissolved in 32 μ L dissolving damping fluid.
1.3DNA fragment 3 ' end adds " A " base
According to following proportioning ready reaction mixture:
DNA 32 μ L after terminal the reparation
Klenow enzyme buffer liquid 5 μ L
dATP(1mM) 10μL
The Klenow enzyme (3 ' to 5 ' 5 prime excision enzyme activity) 3 μ L
The comfort type constant-temperature mixer is transferred to 37 ℃, and then reaction 30min carries out purifying with MiniElute PCR purification kit, at last sample is dissolved in 10 μ L Elution Buffer.
1.4 connect DNA label joint
According to following proportioning ready reaction mixture:
The DNA 10 μ L that above-mentioned steps obtains
T4DNA ligase enzyme damping fluid 25 μ L
DNA Index-N joint (takara company is synthetic) 10 μ L
Annotate: for different samples, use different DNA Index-N joints (N=1～67).Employed DNA Index-N joint can be arbitrary DNA label joint to forming after DNA Index-NF_adapter and the DNA Index-NR_adapter annealing shown in the table 2.
(transfer to 20 ℃, then reaction 15min carries out purifying with QIAquick PCR purification kit, at last sample is dissolved in the 30 μ L dissolving damping fluid with the comfort type constant-temperature mixer.
Reclaim purifying 1.5 connect the glue of product
In 2% agarose gel, carry out electrophoretic separation with connecting product; Subsequently the purpose fragment of 280～300bp is put into the Eppendorf pipe.Carry out glue purification with QIAquick glue purification test kit and reclaim, reclaim product and be dissolved in 30 μ L Elution Buffer.
1.6PCR reaction
The PCR reaction: the reaction system ready reaction mixture according to following is positioned over reagent on ice.
Glue reclaims the DNA 10 μ L behind the purifying
PE PCR Primers 1.0 1μL
PE PCR Primers 2.0 1μL
Phusion archaeal dna polymerase 25 μ L
ddH
2O 13μL
The PCR reaction conditions
9g℃30s
72℃5min
4 ℃ of preservations
1.7PCR the glue of product reclaims purifying
With PCR product electrophoretic separation in 2% agarose gel, the purpose fragment of 280～300bp is reclaimed in cutting, carries out glue purification with QIAquick glue purification test kit and reclaims, and reclaims product and is dissolved in 30 μ L Elution Buffer.
1.8DNA the preparation product detects
Use Agilent 2100 Bioanalyzer according to the working method of manufacturer's specification sheets, detect library output (seeing Fig. 6).The library clip size of surveying is 284bp, and concentration is 32.64ng/ul.Library size and concentration are all qualified.
2. interpretation of result
67 DNA tag library electrophoresis result that make up are illustrated in fig. 5 shown below, and have used respectively the marker of D2000 and 50bp among Fig. 5, originate from respectively day root company and NEB company; Arrow institute mark be purpose library clip size.
Solexa sequencing result statistics: it is that 0 erroneous matching (mismatch) accounts for 98.43% that label is identified fully, label measure wrong 1 base namely 1 erroneous matching account for 0.09%, other reading results (other reads) account for 1.48%, so the recognition rate of sequencing result label is 98.5%, can satisfy the order-checking requirement of solexa DNA index.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Reference
1、Paired-End sequencing User Guide；illumina part#1003880
2、Preparing samples for ChIP sequencing for DNA；illumina part#11257047 Rev.A；
3、mRNA sequencing sample preparation Guide；illumina part#1004898 Rev.D
4、Preparing 2-5kb samples for mate pair library sequencing；illumina part#1005363 Rev.B；
5、Preparing samples for multiplexed Paired-End sequencing；illumina part#1005361 Rev.B；
Claims (44)
1. one group of DNA label, described DNA label comprise DNA Index-1 in 67 DNA labels shown in the table 2～DNA Index-5 at least.
2. one group of DNA label claimed in claim 1, it also comprises DNA Index-6 in 67 DNA labels shown in the table 2～DNA Index-10.
3. one group of DNA label claimed in claim 1, it also comprises DNA Index-11 in 67 DNA labels shown in the table 2～DNA Index-15.
4. one group of DNA label claimed in claim 1, it also comprises DNA Index-16 in 67 DNA labels shown in the table 2～DNA Index-20.
5. one group of DNA label claimed in claim 1, it also comprises DNA Index-21 in 67 DNA labels shown in the table 2～DNA Index-25.
6. one group of DNA label claimed in claim 1, it also comprises DNA Index-26 in 67 DNA labels shown in the table 2～DNA Index-30.
7. one group of DNA label claimed in claim 1, it also comprises DNA Index-31 in 67 DNA labels shown in the table 2～DNA Index-35.
8. one group of DNA label claimed in claim 1, it also comprises DNA Index-36 in 67 DNA labels shown in the table 2～DNA Index-40.
9. one group of DNA label claimed in claim 1, it also comprises DNA Index-41 in 67 DNA labels shown in the table 2～DNA Index-45.
10. one group of DNA label claimed in claim 1, it also comprises DNA Index-46 in 67 DNA labels shown in the table 2～DNA Index-50.
11. one group of DNA label claimed in claim 1, it also comprises DNA Index-51 in 67 DNA labels shown in the table 2～DNA Index-55.
12. one group of DNA label claimed in claim 1, it also comprises DNA Index-56 in 67 DNA labels shown in the table 2～DNA Index-60.
13. one group of DNA label claimed in claim 1, it also comprises DNA Index-61 in 67 DNA labels shown in the table 2～DNA Index-65.
14. one group of DNA label claimed in claim 1, it also comprises DNA Index-63 in 67 DNA labels shown in the table 2～DNA Index-67.
15. each described one group of DNA label is used for the purposes of constructed dna tag library among the claim 1-14, wherein the DNA label joint of DNA tag library comprises described DNA label at 3 ' end, thereby consists of corresponding separately DNA label joint.
16. the described purposes of claim 15, wherein said DNA label joint are used as 5 ' joint and the 3 ' joint of DNA tag library simultaneously.
17. the described purposes of claim 15, wherein said DNA label are inserted in the 3 ' end in the DNA label joint, perhaps by or be not connected to 3 ' end of DNA joint by connexon.
18. the described purposes of claim 17, wherein said DNA label are not connected to 3 ' end of DNA joint by connexon.
19. one group of DNA label joint, wherein the DNA label joint of DNA tag library comprises each described label among the claim 1-14 at 3 ' end, and described DNA label joint comprises DNA Index-1F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-5F/R_adapter at least.
20. the described one group of DNA label joint of claim 19, it also comprises DNA Index-6F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-10F/R_adapter.
21. the described one group of DNA label joint of claim 19, it also comprises DNA Index-11F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-15F/R_adapter.
22. the described one group of DNA label joint of claim 19, it also comprises DNA Index-16F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-20F/R_adapter.
23. the described one group of DNA label joint of claim 19, it also comprises DNA Index-21F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-25F/R_adapter.
24. the described one group of DNA label joint of claim 19, it also comprises DNA Index-26F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-30F/R_adapter.
25. the described one group of DNA label joint of claim 19, it also comprises DNA Index-31F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-35F/R_adapter.
26. the described one group of DNA label joint of claim 19, it also comprises DNA Index-36F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-40F/R_adapter.
27. the described one group of DNA label joint of claim 19, it also comprises DNA Index-41F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-45F/R_adapter.
28. the described one group of DNA label joint of claim 19, it also comprises DNA Index-46F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-50F/R_adapter.
29. the described one group of DNA label joint of claim 19, it also comprises DNA Index-51F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-55F/R_adapter.
30. the described one group of DNA label joint of claim 19, it also comprises DNA Index-56F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-60F/R_adapter.
31. the described one group of DNA label joint of claim 19, it also comprises DNA Index-61F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-65F/R_adapter.
32. the described one group of DNA label joint of claim 19, it also comprises DNA Index-66F/R_adapte in 67 DNA label joints shown in the table 2～DNA Index-67F/R_adapter.
33. each described one group of DNA label joint among the claim 19-32, wherein said DNA label joint are simultaneously as 5 ' joint and 3 ' joint.
34. each described one group of DNA label joint is used for the purposes of constructed dna tag library among the claim 19-32.
35. the described purposes of claim 34, wherein said DNA label joint are used as 5 ' joint and the 3 ' joint of DNA tag library simultaneously.
36. the DNA tag library by each described one group of DNA label joint structure among the claim 19-32.
37. the construction process of a tag library, it comprises:
1) provide n DNA sample, n is the integer of integer and 1≤n≤67, and described DNA sample includes but not limited to people DNA sample from all eucaryons and procaryotic DNA sample;
2) human gene group DNA is interrupted, the method that wherein interrupts includes but not limited to that ultrasonic wave interrupts method;
3) the terminal reparation;
4) dna fragmentation 3 ' end adds " A " base;
5) connect DNA label joint;
6) with step 5) the connection product that obtains carries out gel purification, and the recovery product of each DNA sample mixed;
7) step 6 is used in PCR reaction) the mixture of recovery product as template, under the condition of purpose nucleic acid that is suitable for increasing, carry out pcr amplification, the PCR product is carried out glue reclaims purifying;
Wherein said DNA label joint is as among the claim 19-32 as described in each.
38. the described method of claim 37, wherein step 1) in, n is integer and 2≤n≤67.
39. the described method of claim 37, wherein step 2) in, the DNA band after interrupting is concentrated on about 180bp.
40. the described method of claim 37, wherein step 5) in, each label joint is connected to the two ends of dna fragmentation.
41. the described method of claim 37, wherein step 6) in, the connection product carries out electrophoresis and recovery by 2% agarose gel.
42. the described method of claim 37, wherein step 7) in, the purpose fragment of recovery 280～300bp.
43. the described method of claim 37, wherein step 7) primer that uses in the PCR reaction comprises
PE PCR Primers1.0：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT CT; With
PE PCR Primers2.0：
CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT。
44. the tag library by the described method structure of claim 37.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010299257 CN102409045B (en) | 2010-09-21 | 2010-09-21 | Tag library constructing method based on DNA (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter |
| PCT/CN2011/079898 WO2012037876A1 (en) | 2010-09-21 | 2011-09-20 | Dna tag and application thereof |
| HK12109359.2A HK1168626A1 (en) | 2010-09-21 | 2012-09-24 | Tag library constructing method based on dna (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010299257 CN102409045B (en) | 2010-09-21 | 2010-09-21 | Tag library constructing method based on DNA (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102409045A CN102409045A (en) | 2012-04-11 |
| CN102409045B true CN102409045B (en) | 2013-09-18 |
Family
ID=45873441
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010299257 Active CN102409045B (en) | 2010-09-21 | 2010-09-21 | Tag library constructing method based on DNA (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN102409045B (en) |
| HK (1) | HK1168626A1 (en) |
| WO (1) | WO2012037876A1 (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374759B (en) * | 2012-04-26 | 2015-09-30 | 中国科学院上海生命科学研究院 | A kind of detection of lung cancer shifts method and the application thereof of significant SNP |
| CN103571822B (en) * | 2012-07-20 | 2016-03-30 | 中国科学院植物研究所 | A kind of multipurpose DNA fragmentation enriching method analyzed for new-generation sequencing |
| CN102952877B (en) * | 2012-08-06 | 2014-09-24 | 深圳华大基因研究院 | Method and system for detecting alpha-globin gene copy number |
| CN103290104B (en) * | 2013-01-23 | 2016-03-02 | 北京诺禾致源生物信息科技有限公司 | A kind of genomic samples breaking method being applied to the simple and direct cheapness of s-generation order-checking |
| US10302614B2 (en) | 2014-05-06 | 2019-05-28 | Safetraces, Inc. | DNA based bar code for improved food traceability |
| JP6483518B2 (en) * | 2014-05-20 | 2019-03-13 | 信越化学工業株式会社 | Conductive polymer composite and substrate |
| CN107075513B (en) | 2014-09-12 | 2020-11-03 | 深圳华大智造科技有限公司 | Isolated oligonucleotides and their use in nucleic acid sequencing |
| WO2016049929A1 (en) * | 2014-09-30 | 2016-04-07 | 天津华大基因科技有限公司 | Method for constructing sequencing library and application thereof |
| US10962512B2 (en) | 2015-08-03 | 2021-03-30 | Safetraces, Inc. | Pathogen surrogates based on encapsulated tagged DNA for verification of sanitation and wash water systems for fresh produce |
| CN105506125B (en) * | 2016-01-12 | 2019-01-22 | 上海美吉生物医药科技有限公司 | A kind of sequencing approach and a kind of two generation sequencing libraries of DNA |
| CN105734048A (en) * | 2016-02-26 | 2016-07-06 | 武汉冰港生物科技有限公司 | PCR-free sequencing library preparation method for genome DNA |
| CN108728903A (en) * | 2017-04-21 | 2018-11-02 | 深圳市乐土精准医疗科技有限公司 | The banking process of thalassemia large sample screening is used for based on high-flux sequence |
| CN108949905B (en) * | 2017-05-23 | 2022-05-17 | 深圳华大基因股份有限公司 | Control library and construction method thereof |
| US10926264B2 (en) | 2018-01-10 | 2021-02-23 | Safetraces, Inc. | Dispensing system for applying DNA taggants used in combinations to tag articles |
| US11853832B2 (en) | 2018-08-28 | 2023-12-26 | Safetraces, Inc. | Product tracking and rating system using DNA tags |
| US11200383B2 (en) | 2018-08-28 | 2021-12-14 | Safetraces, Inc. | Product tracking and rating system using DNA tags |
| CN110468188B (en) * | 2019-08-22 | 2023-08-22 | 广州微远医疗器械有限公司 | Tag sequence set for second generation sequencing and design method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005068656A1 (en) * | 2004-01-12 | 2005-07-28 | Solexa Limited | Nucleic acid characterisation |
| CN101100764A (en) * | 2007-06-13 | 2008-01-09 | 北京万达因生物医学技术有限责任公司 | Molecule substitution label sequencing parallel detection method-oligomictic nucleic acid coding label molecule library micro-sphere array analysis |
| WO2008093098A2 (en) * | 2007-02-02 | 2008-08-07 | Illumina Cambridge Limited | Methods for indexing samples and sequencing multiple nucleotide templates |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK1991698T3 (en) * | 2006-03-01 | 2014-03-10 | Keygene Nv | "High-throughput" -sekvensbaseret detection of SNPs using ligeringsassays |
| CN101434988B (en) * | 2007-11-16 | 2013-05-01 | 深圳华因康基因科技有限公司 | High throughput oligonucleotide sequencing method |
| US8236503B2 (en) * | 2008-11-07 | 2012-08-07 | Sequenta, Inc. | Methods of monitoring conditions by sequence analysis |
| CN101748213B (en) * | 2008-12-12 | 2013-05-08 | 深圳华大基因研究院 | Environmental microorganism detection method and system |
-
2010
- 2010-09-21 CN CN 201010299257 patent/CN102409045B/en active Active
-
2011
- 2011-09-20 WO PCT/CN2011/079898 patent/WO2012037876A1/en active Application Filing
-
2012
- 2012-09-24 HK HK12109359.2A patent/HK1168626A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005068656A1 (en) * | 2004-01-12 | 2005-07-28 | Solexa Limited | Nucleic acid characterisation |
| WO2008093098A2 (en) * | 2007-02-02 | 2008-08-07 | Illumina Cambridge Limited | Methods for indexing samples and sequencing multiple nucleotide templates |
| CN101100764A (en) * | 2007-06-13 | 2008-01-09 | 北京万达因生物医学技术有限责任公司 | Molecule substitution label sequencing parallel detection method-oligomictic nucleic acid coding label molecule library micro-sphere array analysis |
Also Published As
| Publication number | Publication date |
|---|---|
| HK1168626A1 (en) | 2013-01-04 |
| WO2012037876A1 (en) | 2012-03-29 |
| CN102409045A (en) | 2012-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102409045B (en) | Tag library constructing method based on DNA (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter | |
| CN102409048B (en) | DNA index library building method based on high throughput sequencing | |
| CN102409049B (en) | DNA(deoxyribonucleic acid) index library building method based on PCR (polymerase chain reaction) | |
| CN101967476B (en) | Joint connection-based deoxyribonucleic acid (DNA) polymerase chain reaction (PCR)-free tag library construction method | |
| EP3211100A1 (en) | Amplification primers and methods | |
| US10718015B2 (en) | Sequencing library, preparation method and use thereof | |
| CN105986015B (en) | Method and kit for detecting one or more target sequences of multiple samples based on high-throughput sequencing | |
| CN104562213A (en) | Amplification sublibrary and construction method thereof | |
| CN103060924A (en) | Library preparation method of trace nucleic acid sample and application thereof | |
| CN102690809B (en) | DNA index and application thereof in construction and sequencing of mate-paired indexed library | |
| CN102409046B (en) | Small RNA (ribonucleic acid) tags | |
| CN102653784A (en) | Tag used for multiple nucleic acid sequencing and application method thereof | |
| CN105734048A (en) | PCR-free sequencing library preparation method for genome DNA | |
| CN109321567A (en) | Sequencing DNA library kit and sequencing DNA library construction method | |
| CN104153003A (en) | Method for establishing DNA (Deoxyribose Nucleic Acid) library based on illumina sequencing platform | |
| CN102978206A (en) | High-throughput sequencing joint applied to hybrid library building and library building method thereof | |
| CN105401222A (en) | Method for constructing DNA library used for sequencing | |
| CN102409043B (en) | Method for constructing high-flux and low-cost Fosmid library, label and label joint used in method | |
| CN106676099B (en) | Construct the method and kit for simplifying genomic library | |
| CN105441429A (en) | Adenylylation joint preparation method and adenylylation joint | |
| CN115386622B (en) | Library construction method of transcriptome library and application thereof | |
| CN108342385A (en) | A kind of connector and the method that sequencing library is built by way of high efficiency cyclisation | |
| CN107794258A (en) | A kind of method and its application in constructed dna large fragment library | |
| CN112251491B (en) | cDNA library construction method of capillary 96-well plate | |
| CN107058300A (en) | The tape label joint built for single-molecule sequencing library template and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1168626 Country of ref document: HK |
|
| ASS | Succession or assignment of patent right |
Owner name: BGI TECHNOLOGY SOLUTIONS CO., LTD. Free format text: FORMER OWNER: BGI-SHENZHEN CO., LTD. Effective date: 20130715 Free format text: FORMER OWNER: BGI-SHENZHEN Effective date: 20130715 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20130715 Address after: 518083 science and Technology Pioneer Park, comprehensive building, Beishan Industrial Zone, Yantian District, Guangdong, Shenzhen 201 Applicant after: BGI Technology Solutions Co., Ltd. Address before: Beishan Industrial Zone Building in Yantian District of Shenzhen city of Guangdong Province in 518083 Applicant before: BGI-Shenzhen Co., Ltd. Applicant before: BGI-Shenzhen |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1168626 Country of ref document: HK |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190726 Address after: 214200 Xingye Road, Xinjie Street, Yixing City, Wuxi City, Jiangsu Province, 298 Patentee after: Huada Qinglan Biotechnology (Wuxi) Co., Ltd. Address before: 518083 science and Technology Pioneer Park, comprehensive building, Beishan Industrial Zone, Yantian District, Guangdong, Shenzhen 201 Patentee before: BGI Technology Solutions Co., Ltd. |