CN107988219B - Catenation sequence and its design method for multiplex PCR library construction - Google Patents
Catenation sequence and its design method for multiplex PCR library construction Download PDFInfo
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Abstract
The invention discloses the catenation sequences and its design method for multiplex PCR library construction.Catenation sequence based on the present invention can realize that the connection of label, sequence measuring joints, target fragment, design method include by PCR amplification：Obtain candidate sequence collection, candidate sequence primary dcreening operation, e-PCR analyses；The catenation sequence of the present invention is eliminated to be interfered with each other with other sequences in amplification system, the Library Quality of structure is good, and it is versatile, it is not only adapted to a step and library is built in the amplification of two steps, it is further adapted for the structure of multiple nucleic acids sequencing library, such as detect hereditary hearing impairment pathogenic sites, phenylketonuria Disease-causing gene, lung cancer targets medication gene, breast cancer BRCA1/2 mutators, the sequencing library of safe medication gene, library construction based on catenation sequence simplifies Library development flow, greatly save sequencing cost, it applies also in nucleic acid sequencing kit, with very good market application prospect.
Description
Technical field
The invention belongs to high-flux sequence field, more particularly relate to multiplex PCR library construction catenation sequence and
Its design method.
Background technology
Nucleic acid sequencing have become in biological study, disorder in screening, medicine auxiliary diagnosis indispensable one it is important
Technology fundamentally changes the mode of human research's life blueprint.High-flux sequence is the method for nucleic acid sequencing of current mainstream,
It is low with expense, flux is big, fireballing advantage.Existing high-flux sequence platform includes mainly Illumina companies
Solexa microarray datasets, 454 platforms of Roche companies, Life companies Ion torrent platforms.
With the promotion of microarray dataset hardware and software, hinder high throughput sequencing technologies development be library construction and
Subsequent data analysis.Wherein, all microarray datasets are directed to complicated library construction process, on the one hand need to capture target patch
On the other hand section also needs to the label of the mating sequence measuring joints of platform and differentiation sample being connected in capture segment.To be based on
For the library construction process of multiplex PCR capture, basic methods include the following steps：(1) multiplex PCR captures target fragment；(2)
Primer and/or modification are removed；(3) sequence measuring joints and/or label connection；(4) magnetic beads for purifying；(5) PCR amplification is enriched with.In this base
The improvement made in plinth flow mainly has：(1) connection of sequence measuring joints and label merges into a step with PCR amplification；(2) multiple
PCR captures target fragment is connect with sequence measuring joints and/or label merges into a step；(3) step is removed in primer free and/or modification,
(4) combination etc. of case above, main purpose is all simple flow, cost-effective.The simplification of library construction process is not
Easy thing, the improvement or removal of each step can influence the quality of library construction, such as library concentration, and library fragments are distributed, in turn
Influence amplicon homogeneity, data user rate etc..In addition, more can not be ignored, in PCR sequencing library building process, it is related to
Reaction sequence complexity it is various, such as specific primer sequence, sequence measuring joints sequence, multiple sequence labels, how in Jian Ku
Interfering with each other between exclusion or reduction sequence, obtains good Library Quality, and take into account the versatility of sequence, is in step
Designer is needed to go design and improvement that could realize creatively.
Invention content
The technical issues of being referred to based on background technology, the purpose of the present invention is to provide one kind being used for multiplex PCR library structure
The catenation sequence and its design method built；The above catenation sequence and the connection primer constituted based on catenation sequence, connection are also provided
The application of connector, connection label in nucleic acid sequencing library construction and in preparing nucleic acid sequencing kit.
The technical solution used in the present invention is：
A kind of catenation sequence for multiplex PCR library construction, the catenation sequence are set to target fragment specificity and draw
5 ' ends of object, constitute connection primer；The catenation sequence is also provided at 3 ' ends of sequence measuring joints, constitutes jointing；Based on even
The connection of target fragment and sequence measuring joints is realized in the setting for connecing sequence by PCR amplification.
As the improvement of above-mentioned catenation sequence, the catenation sequence may also be disposed on 3 ' ends of label, constitute connection label,
The connection of label, sequence measuring joints, target fragment is realized by PCR amplification.
As the preferred of above-mentioned catenation sequence, the catenation sequence is selected from SEQ ID NO:1~SEQ ID NO:7 is any one
Nucleotide sequence.
As the preferred of above-mentioned catenation sequence, the catenation sequence is used for Ion torrent microarray datasets.
As the preferred of above-mentioned catenation sequence, the catenation sequence is suitable for people's nucleic acid sequencing library construction.
The design method of catenation sequence as described above, includes the following steps：
Obtain candidate sequence collection：Using the remote source species gene group nucleotide sequence collection N of the sliding window method structure mankind and mankind's base
Because of a group nucleotide sequence collection M, the sequence occurred in sequence sets M is rejected from sequence sets N, obtains candidate sequence collection；
Candidate sequence primary dcreening operation：It is concentrated in candidate sequence, screening meets the sequence of the following conditions：
(1) sequence that melting temperature is 55~65 DEG C；
(2) sequence of unknown base is not included；
(3) sequence of dimer is not formed with sequence itself, sequence measuring joints；
E-PCR secondary screenings：Sequence pair mankind's reference gene group that primary dcreening operation is obtained carries out e-PCR analyses, and screening does not generate mould
The sequence of quasi- amplified production is as catenation sequence.
As the preferred of above-mentioned design method, when obtaining candidate sequence collection, sliding window method using 15~20bp as window size,
1bp is step-length, scans corresponding reference gene group.
As the preferred of above-mentioned design method, screening does not form the side of the sequence of dimer with sequence itself, sequence measuring joints
Method is：Using primer softwares, each sequence that candidate sequence is concentrated carries out dimer with sequence itself, sequence measuring joints respectively
The sequence that can form dimer is rejected in analysis.
As the preferred of above-mentioned design method, the remote source species of the mankind include that bacterium, fungi, plant, the remote source of the mankind are dynamic
Object；The remote source species of the mankind at least select a species.
Catenation sequence, connection primer, jointing, connection label are in nucleic acid sequencing library construction and preparation as described above
Application in nucleic acid sequencing kit.
The beneficial effects of the invention are as follows：
Based on the catenation sequence of the present invention, the connection of label, sequence measuring joints, target fragment can be realized by PCR amplification,
Design method includes：Obtain candidate sequence collection, candidate sequence primary dcreening operation, e-PCR secondary screenings；The catenation sequence and amplification system of the present invention
Interfering with each other for middle other sequences is small, and the Library Quality of structure is good and versatile, is not only adapted to a step and the amplification of two steps is built
Library is further adapted for the structure of multiple nucleic acids sequencing library, such as detect hereditary hearing impairment pathogenic sites, phenylketonuria Disease-causing gene,
Lung cancer targets the nucleic acid sequencing library of medication gene, breast cancer BRCA1/2 mutators, safe medication gene, based on connection sequence
The nucleic acid sequencing library construction of row simplifies Library development flow, and sequencing cost is greatly saved, and applies also for nucleic acid sequencing reagent
In box, there is very good market application prospect.
In addition, effect of the inventor based on catenation sequence, the creative design method for proposing a set of catenation sequence is comprehensive
It closes and considers interaction between sequence length, melting temperature, sequence, the factor with human genome mutual exclusion etc., by this
Design method, can filter out specific height, and versatile catenation sequence realizes catenation sequence in nucleic acid sequencing library construction
And the application in nucleic acid sequencing kit.
Description of the drawings
Fig. 1 is the segment distribution in the hereditary hearing impairment pathogenic sites library of different catenation sequence structures, wherein figure A~G bases
In catenation sequence U1~U7；
Fig. 2 is the box figure of amplicon homogeneity in the hereditary hearing impairment pathogenic sites library of catenation sequence U4 structures, wherein
Abscissa is amplicon, and ordinate is reads numbers；
Fig. 3 is the sequencing peak figure for the phenylketonuria mutant gene libraries that embodiment 3 is built；
Fig. 4 is the box figure of amplicon homogeneity for the phenylketonuria mutant gene libraries that embodiment 3 is built, wherein horizontal seat
It is designated as amplicon, ordinate is reads numbers；
Fig. 5 is the sequencing peak figure for the lung cancer targeting medication gene library that embodiment 4 is built；
Fig. 6 is the box figure of amplicon homogeneity for the lung cancer targeting medication gene library that embodiment 4 is built, wherein abscissa
For amplicon, ordinate is reads numbers；
Fig. 7 is the sequencing peak figure for the breast cancer BRCA1/2 mutant gene libraries that embodiment 5 is built；
Fig. 8 is the box figure of amplicon homogeneity for the breast cancer BRCA1/2 mutant gene libraries that embodiment 5 is built, wherein
Abscissa is amplicon, and ordinate is reads numbers；
Fig. 9 is the sequencing peak figure for the safe medication gene library that embodiment 6 is built；
Figure 10 is the box figure of amplicon homogeneity for the safe medication gene library that embodiment 6 is built, and wherein abscissa is
Amplicon, ordinate are reads numbers.
Specific implementation mode
A kind of catenation sequence for multiplex PCR library construction, the catenation sequence are set to target fragment specificity and draw
5 ' ends of object, constitute connection primer；The catenation sequence is also provided at 3 ' ends of sequence measuring joints, constitutes jointing；Based on even
The connection of target fragment and sequence measuring joints is realized in the setting for connecing sequence by PCR amplification；Further, the catenation sequence
3 ' the ends that may also be disposed on label, constitute connection label, pass through PCR amplification realization label, the company of sequence measuring joints, target fragment
It connects；The optional one-step or two-step amplification of amplification, but not limited to this.
Preferably, the catenation sequence is selected from SEQ ID NO:1~SEQ ID NO:7 any one nucleotides sequences
Row.
It is further preferred that the catenation sequence is used for Ion torrent microarray datasets.
It is further preferred that the catenation sequence is suitable for people's nucleic acid sequencing library construction.
Wherein, target fragment specific primer, sequence measuring joints and label belong to the common knowledge of this field, and target fragment is special
Specific primer can be designed according to techniques well known and be obtained, such as oligo primer-design softwares；Sequence measuring joints are flat according to sequencing
The adaptability of platform selects, and A connectors and P connectors as utilized Ion torrent microarray datasets in embodiment, label can utilize ability
Conventional tags well known to domain, 96 labels (Barcode01~Barcode96) provided such as Life platforms.
It, can be by library construction process simplification, by one-step or two-step PCR amplification, i.e., based on " catenation sequence " of the present invention
It can get sequencing library.Specifically, the Library development flow of two-step method is without removing primer and/or modification, by sequence measuring joints and label
The enrichment of connection and PCR amplification merge into a step, it is a step that multiplex PCR, which captures target fragment separately,；Preferably, one-step method can be two
On the basis of footwork Library development flow, connection, the PCR amplification enrichment of multiplex PCR capture target fragment, sequence measuring joints and label are realized
Three steps are unified, and time cost and economic cost is greatly saved.
The design method of catenation sequence of the present invention, includes the following steps：
Obtain candidate sequence collection：Using the remote source species gene group nucleotide sequence collection N of the sliding window method structure mankind and mankind's base
Because of a group nucleotide sequence collection M, the sequence occurred in sequence sets M is rejected from sequence sets N, obtains candidate sequence collection.
Wherein, it is contemplated that sequence length is too short, is difficult to meet primer attribute of the catenation sequence in amplification, sequence length
It is long, influence the read length that sequencing generates；When obtaining candidate sequence collection, sliding window method is using 15~20bp as window size, 1bp
For step-length, corresponding reference gene group is scanned, corresponding sequence sets are obtained.
Wherein, the remote source species of the mankind include bacterium, fungi, plant, the remote source animal of the mankind, and remote source species at least select one
Species, but not limited to this.
Candidate sequence primary dcreening operation：It is concentrated in candidate sequence, screening meets the sequence of the following conditions：
(1) sequence that melting temperature is 55~65 DEG C；
(2) sequence of unknown base is not included；
(3) sequence of dimer is not formed with sequence itself, sequence measuring joints.
Wherein, the specificity of catenation sequence is considered in the selection of melting temperature, and need to be with other sequences in same system
Effect；
Wherein, catenation sequence should avoid the hybridization between sequence complementary, and screening does not form two with sequence itself, sequence measuring joints
When the method for the sequence of aggressiveness：Using primer softwares, each sequence that candidate sequence is concentrated respectively with sequence itself, sequencing
Connector carries out dimer assay, rejects the sequence that can form dimer.
E-PCR secondary screenings：Sequence pair mankind's reference gene group that primary dcreening operation is obtained carries out e-PCR analyses, and screening does not generate mould
The sequence of quasi- amplified production is as catenation sequence.
As previously mentioned, the catenation sequence of the present invention, the connection primer, jointing, the connection mark that are constituted based on catenation sequence
Application of the label in nucleic acid sequencing library construction and in preparing nucleic acid sequencing kit.
Present invention will be further explained below with reference to specific examples, and target fragment specific primer that embodiment is related to is surveyed
Sequence connector and label can be obtained according to routine techniques, not repeated；Unspecified experimental method and condition in embodiment
It presses this field routine or manufacturer's recommendations operates.
Embodiment 1, catenation sequence
Using catenation sequence design method provided by the invention, the remote source species of the mankind refer to following species Exophiala
mesophila、Photobacterium profundum、Borrelia miyamotoi、Neomicrococcus
aestuarii；Mankind's reference gene group is hg19；The A connectors and P that sequence measuring joints are provided by Ion torrent microarray datasets connect
Head；Finishing screen selects 7 catenation sequences, nucleotide sequence such as SEQ ID NO:1~SEQ ID NO:Shown in 7.
U1：5'-GGGCTGGCAAGCCACGTT-3'(SEQ ID NO:1)；
U2：5'-GTCTCGTGGGCTCGGAGA-3'(SEQ ID NO:2)；
U3：5'-CGTCGCCGTCCAGCTCGA-3'(SEQ ID NO:3)；
U4：5'-AAATGGGCGGTAGGCTTG-3'(SEQ ID NO:4)；
U5：5'-CGTGACGCGGTGCAGGGC-3'(SEQ ID NO:5)；
U6：5'-CGCAAATGGGCGGTAGGC-3'(SEQ ID NO:6)；
U7：5'-CCGGGAGCTGCATGTGTC-3'(SEQ ID NO:7).
The application of embodiment 2, different catenation sequences in hereditary hearing impairment pathogenic sites sequencing library structure
By taking the detection of hereditary hearing impairment pathogenic sites as an example, it is based respectively on the thinking structure of the catenation sequence and the present invention of U1~U7
Build nucleic acid sequencing library；By 41 couples of 0.2 μ L of connection primer mixture, 0.5 μ L of jointing, 0.5 μ L of connection label, sample gene
Group DNA 20ng, 12.5 μ L of PCR reaction solution mix, no enzyme water are mended to 25 μ L, and step amplification obtains sequencing library, and amplification condition is
95 DEG C of pre-degeneration 3min；35 cyclic amplifications (95 DEG C of denaturation 15s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s)；72 DEG C of extensions
1min, 16 DEG C of forever；Amplified production carries out high-flux sequence through magnetic beads for purifying, using Ion Torrent microarray datasets.
Agilent 2100 analyzes the segment of sequencing library, and the results are shown in Figure 1, and all catenation sequences can
Amplifying target fragment, wherein the Library Quality based on catenation sequence U3, U4, U6 structure is more excellent, and non-specific amplification is less,
It is furthermore preferred that the Library Quality based on U4 structures is best, target fragment peak area is maximum, in addition, with reference to Fig. 2, amplicon is equal
One property is good.In sequencing data, the results are shown in Table 1 for the long ratio and data user rate less than 30bp of reading, consistent, it is based on connecting
The Library Quality of sequence U3, U4, U6 structure is more excellent, and data user rate is high, and reads long relatively low less than the ratio of 30bp, wherein
Library based on U4 structures is optimal.
Result above illustrates that the catenation sequence based on the present invention can realize the library of building in nucleic acid sequencing library, further
, it may be implemented to complete step amplification under comprising a variety of sequence systems such as template, specific primer, sequence measuring joints, label,
Middle catenation sequence U4 is optimal, is accordingly used in subsequent applications.
Table 1, different catenation sequence effect compare
| Sequence | Read the long ratio less than 30bp | Data user rate | It evaluates in library |
| U1 | 17.3% | 42.2% | Generally |
| U2 | 25.5% | 32.8% | It is poor |
| U3 | 4.6% | 69.5% | It is excellent |
| U4 | 3.8% | 74.8% | It is optimal |
| U5 | 15.5% | 48.8% | Generally |
| U6 | 7.2% | 60.5% | It is excellent |
| U7 | 20.1% | 40.2% | It is poor |
The application of embodiment 3, catenation sequence U4 in phenylketonuria Disease-causing gene sequencing library structure
Thinking of the present invention is pressed by taking the detection of phenylketonuria Disease-causing gene as an example using catenation sequence U4, is expanded using two steps
Method：By 61 pairs of 2 μ L of connection primer mixture, sample gene group DNA 200ng, 10 μ L of PCR reaction solution mix, 1 DMSO μ L, nothings
Enzyme water is mended to 20 μ L, and first step PCR amplification is carried out, and realizes the connection of catenation sequence and target fragment；First step pcr amplification reaction
Condition setting is as follows：95 DEG C of pre-degeneration 5min；(95 DEG C of denaturation 35s, 60 DEG C of annealing 90s, 72 DEG C extend 15 cyclic amplifications
30s), extend 1min, 16 DEG C of forever for 72 DEG C；Then by after magnetic beads for purifying 10.5 μ L of first step pcr amplification reaction product,
0.5 μ L of jointing, 0.5 μ L of connection label, 1 DMSO μ L, 12.5 μ L mixing of PCR reaction solution mix, carry out second step PCR expansions
Increase reaction, obtains sequencing library；Amplification condition is 98 DEG C of pre-degeneration 3min；(98 DEG C of denaturation 20s, 60 DEG C are moved back 25 cyclic amplifications
Fiery 60s, 72 DEG C of extension 30s), 72 DEG C extend 1min, 16 DEG C of forever；Amplified production is through magnetic beads for purifying, using Ion
Torrent microarray datasets carry out high-flux sequence.
It is 18.9ng/ μ L that library, which quantitatively detects library concentration, upper machine sequencing peak figure as shown in figure 3, non-specific amplification is few,
Library Quality is high；Sequencing data is also shown：Data user rate 76.5% compares success rate 84.6%, sequencing coverage rate reaches
100%；A large amount of detection datas show that with reference to Fig. 4, amplicon homogeneity is high, it is seen that catenation sequence of the invention is also applied for benzene
Acetonuria Disease-causing gene sequencing library is built.
The application of embodiment 4, catenation sequence U4 in lung cancer targets medication gene sequencing library construction
Using catenation sequence U4, by taking lung cancer targets medication genetic test as an example, thinking of the present invention is pressed, is expanded using two steps
Method：By 28 couples of connection primer mixture 2 μ L, sample gene group DNA 20ng, 12.5 μ L of PCR reaction solution mix, without enzyme water mend to
25 μ L carry out first step PCR amplification, realize the connection of catenation sequence and target fragment；First step pcr amplification reaction condition setting
It is as follows：95 DEG C of pre-degeneration 3min；15 cyclic amplifications (95 DEG C of denaturation 20s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s), 72 DEG C are prolonged
Stretch 1min, 16 DEG C of forever；Then by 11.5 μ L of first step pcr amplification reaction product, 0.5 μ of jointing after magnetic beads for purifying
L, 0.5 μ L of connection label, 12.5 μ L mixing of PCR reaction solution mix, carry out second step pcr amplification reaction, obtain sequencing library；Expand
Increasing condition is 98 DEG C of pre-degeneration 2min；25 cyclic amplifications (98 DEG C of denaturation 10s, 60 DEG C of annealing 60s, 72 DEG C of extension 30s), 72 DEG C
Extend 1min, 16 DEG C of forever；Amplified production carries out high pass measurement through magnetic beads for purifying, using Ion Torrent microarray datasets
Sequence.
It is 15.8ng/ μ L that library, which quantitatively detects library concentration, upper machine sequencing peak figure as shown in figure 5, non-specific amplification compared with
Few, Library Quality is high；Sequencing data is also shown：Data user rate 64.5% compares success rate 99.7%, sequencing coverage rate reaches
100%；A large amount of detection datas show that with reference to Fig. 6, amplicon homogeneity is high, it is seen that catenation sequence of the invention is also applied for lung
Cancer targets medication gene sequencing library construction.
The application of embodiment 5, catenation sequence U4 in breast cancer BRCA1/2 mutator sequencing libraries structure
Using catenation sequence U4, by taking breast cancer BRCA1/2 abrupt climatic changes as an example, thinking of the present invention is pressed, is expanded using two steps
Method：First by 316 couples of connection primer mixture 2 μ L, sample gene group DNA 20ng, 12.5 μ L of PCR reaction solution mix, without enzyme water
It mends to 25 μ L, carries out first step PCR amplification, realize the connection of catenation sequence and target fragment；First step pcr amplification reaction condition
Setting is as follows：95 DEG C of pre-degeneration 3min；15 cyclic amplifications (95 DEG C of denaturation 20s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s), 72
DEG C extend 1min, 16 DEG C of forever；Then by 11.5 μ L of first step pcr amplification reaction product, the jointing after magnetic beads for purifying
0.5 μ L, 0.5 μ L of connection label, 12.5 μ L mixing of PCR reaction solution mix, carry out second step pcr amplification reaction, obtain sequencing text
Library；Amplification condition is 98 DEG C of pre-degeneration 2min；(98 DEG C of denaturation 10s, 60 DEG C of annealing 60s, 72 DEG C extend 25 cyclic amplifications
30s), extend 1min, 16 DEG C of forever for 72 DEG C；Amplified production is carried out through magnetic beads for purifying using Ion Torrent microarray datasets
High-flux sequence.
It is 16.9ng/ μ L that library, which quantitatively detects library concentration, upper machine sequencing peak figure as shown in fig. 7, non-specific amplification is few,
Library Quality is high；Sequencing data is also shown：Data user rate 56.6% compares success rate 96.7%, sequencing coverage rate reaches
100%；A large amount of detection datas show that with reference to Fig. 8, amplicon homogeneity is high, it is seen that catenation sequence of the invention is also applied for breast
Gland cancer BRCA1/2 mutator sequencing libraries are built.
The application of embodiment 6, catenation sequence U4 in safe medication gene sequencing library construction
Using catenation sequence U4, by taking safe medication genetic test as an example, thinking of the present invention is pressed, 90 pairs of connection primers are mixed
0.6 μ L of object, 0.25 μ L of jointing, 2.5 μ L of connection label, sample gene group DNA 200ng, 12.5 μ L of PCR reaction solution mix,
No enzyme water is mended to 25 μ L, and step amplification obtains sequencing library, and amplification condition is 95 DEG C of pre-degeneration 3min；35 cyclic amplifications (95
DEG C denaturation 10s, 60 DEG C annealing 90s, 72 DEG C extension 30s)；72 DEG C extend 1min, 16 DEG C of forever；Amplified production is pure through magnetic bead
Change, high-flux sequence is carried out using Ion Torrent microarray datasets.
It is 12.3ng/ μ L that library, which quantitatively detects library concentration, upper machine sequencing peak figure as shown in figure 9, non-specific amplification compared with
Few, Library Quality is higher；Sequencing data is also shown：Data user rate 62.5% compares success rate 87.6%, sequencing coverage rate reaches
100%；A large amount of detection datas shows, referring to Fig.1 0, amplicon homogeneity height, it is seen that catenation sequence of the invention is also applied for pacifying
Full medication gene sequencing library construction.
One-step or two-step PCR amplification of the above example based on catenation sequence, effectively constructs detection hereditary hearing impairment
Pathogenic sites, phenylketonuria Disease-causing gene, lung cancer targeting medication gene, breast cancer BRCA1/2 mutators, safe medication base
The nucleic acid sequencing library of cause illustrates that the catenation sequence of the present invention has certain versatility, more confirms the connection sequence of the present invention
Row can be applied to multiple nucleic acids sequencing library structure, be also envisioned that the nucleic acid sequencing examination for being applied to and preparing and being suitable for accordingly detecting
In agent box.In addition, above example is only to illustrate the example of the present invention, protection domain is not limited to this, is not departing from this hair
On the basis of bright catenation sequence and its design method, it is equal that those skilled in the art do not carry out the technical solution that creativity labour obtains
It belongs to the scope of protection of the present invention.
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<213>Artificial sequence ()
<400> 6
cgcaaatggg cggtaggc 18
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 7
ccgggagctg catgtgtc 18
Claims (8)
1. a kind of catenation sequence for multiplex PCR library construction, the catenation sequence is set to target fragment specific primer
5 ' end, constitute connection primer；The catenation sequence is also provided at 3 ' ends of sequence measuring joints, constitutes jointing；Based on connection
The setting of sequence realizes that the connection of target fragment and sequence measuring joints, the catenation sequence are selected from SEQ ID NO by PCR amplification:
1~SEQ ID NO:6 any one nucleotide sequences.
2. the catenation sequence according to claim 1 for multiplex PCR library construction, it is characterised in that：The connection sequence
Row may also be disposed on 3 ' ends of label, constitute connection label, pass through PCR amplification realization label, the company of sequence measuring joints, target fragment
It connects.
3. the catenation sequence according to claim 1 or 2 for multiplex PCR library construction, it is characterised in that：The connection
Sequence is used for Ion torrent microarray datasets.
4. the catenation sequence according to claim 1 or 2 for multiplex PCR library construction, it is characterised in that：The connection
Sequence is suitable for people's nucleic acid sequencing library construction.
5. such as the design method of Claims 1 to 4 any one of them catenation sequence, include the following steps：
Obtain candidate sequence collection：Using the remote source species gene group nucleotide sequence collection N of the sliding window method structure mankind and human genome
Nucleotide sequence collection M rejects the sequence occurred in sequence sets M from sequence sets N, obtains candidate sequence collection；The mankind are remote
Source species are selected fromExophiala mesophila、Photobacterium profundum、Borrelia miyamotoi、 Neomicrococcus aestuarii；
Candidate sequence primary dcreening operation：It is concentrated in candidate sequence, screening meets the sequence of the following conditions：
（1）The sequence that melting temperature is 55~65 DEG C；
（2）Sequence not comprising unknown base；
（3）The sequence of dimer is not formed with sequence itself, sequence measuring joints；
E-PCR secondary screenings：Sequence pair mankind's reference gene group that primary dcreening operation is obtained carries out e-PCR analyses, and screening does not generate simulation and expands
Increase production the sequence of object as catenation sequence.
6. design method according to claim 5, it is characterised in that：When obtaining candidate sequence collection, sliding window method with 15~
20bp is window size, and 1bp is step-length, scans corresponding reference gene group.
7. design method according to claim 5, it is characterised in that：Screening does not form two with sequence itself, sequence measuring joints
The method of the sequence of aggressiveness is：Using primer softwares, each sequence that candidate sequence is concentrated respectively with sequence itself, sequencing
Connector carries out dimer assay, rejects the sequence that can form dimer.
8. Claims 1 to 4 any one of them catenation sequence utilizes 4 any one of them catenation sequence structure of Claims 1 to 4
At connection primer, jointing, connection label people's nucleic acid sequencing library construction and prepare people's nucleic acid sequencing kit in
Using；Wherein, the catenation sequence is set to 5 ' ends of target fragment specific primer, constitutes connection primer；The connection sequence
Row are set to 3 ' ends of sequence measuring joints, constitute jointing；The catenation sequence is set to 3 ' ends of label, constitutes connection mark
Label.
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| Crystal Structure of the Allergen Equ c 1 A DIMERIC LIPOCALIN WITH RESTRICTED IgE-REACTIVE EPITOPES;Marie-Bernard Lascombe等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20000427;第275卷(第28期);第21572–21577页 * |
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