CN106636063A - Primer compound, application thereof and method for establishing library and confirming nucleotide sequence - Google Patents
Primer compound, application thereof and method for establishing library and confirming nucleotide sequence Download PDFInfo
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Abstract
The invention provides a primer compound, an application thereof and a method for establishing a library and confirming a nucleotide sequence. The primer compound comprises a first primer set and a second primer set, wherein the first primer set comprises a first forward primer and a first reverse primer; the first forward primer comprises a target region specific forward primer and a first joint; the first reverse primer comprises a target region specific reverse primer, a reverse library label and a second joint; the second primer set comprises a second forward primer and a second reverse primer; the second forward primer contains a first anneal; the second reverse primer contains a second anneal; the first joint is in annealing complementation with the first anneal in the second forward primer; the second joint is in annealing complementation with the second anneal in the second reverse primer; complete sequencing joints are contained in the products after the PCR amplification of the first joint and the second forward primer as well as the second joint and the second reverse primer.
Description
Technical field
The present invention relates to biomedical sector, specifically, the present invention relates to Primer composition and application thereof, structure treat test sample
The method in product target area nucleic acid sequencing library, the method for determining testing sample target area nucleotide sequence, while determine it is multiple
The method of the target area nucleotide sequence of testing sample.
Background technology
Immunoglobulin, φt cell receptor and HLA etc. are most active molecules in human genome, immune macromole it is many
Sample enables body to recognize and removes countless foreign substances, removes the part metabolite for producing in vivo even apoptosis or decline
Old cell, and remain the balance of body immune system.Immune system be body resist pathogen invasion with it is immunoregulatory
Important system, studies tool and has very important significance to it.Due to the huge multiformity of immune macromole so as to immune macromole
Research full of challenge and difficult.Latest developments are got up and continue welcome immune group storehouse technology and be used to study immune group
Storehouse.The summation at immune group storehouse refers to multifarious immunocyte in an individual a certain moment, and these summations are reacted
The immunoregulation of individual inheritance factor, antigen contact history, surrounding and individuality.Research to immune group storehouse at present concentrates on B
The sequence of cell receptor variable region and the sequence of φt cell receptor variable region, mainly by V gene frameworks area and C areas or J areas
Conservative region design primer enrichment aim sequence, the method for employing includes multiplex PCR, the RACE of arm-PCR and 5 ', then adopts
The method of bioinformatics carries out the analysis of the various dimensions such as sequence length, sequence polymorphism, the sequence similarities and differences to aim sequence.
For at present, to study these huge multifarious regions, it is necessary to by these regions from genome or total
Rna level is enriched with and separates.Multiple PCR method is the Position Design multi-primerses enrichment purpose piece guarded in upstream and downstream
Section, the period for using is usually higher, and the problem that cannot avoid in the enrichment process is exactly multi-primerses in PCR processes
The Preference brought because each pair primer amplification efficiency is inconsistent so that the product section distortion of enrichment, deposits with truth
In certain difference, and it is difficult to correct the distortion of this part from bioinformatics level, it is therefore desirable to take method to reduce
The period of Multiplex PCR.
On the one hand current PCR amplification enrichments target area needs one section of exogenous array of increase between forward and reverse primer
Annealing complementary is realized, cycle-index is more, data volume increases during follow-up sequencing, increased sequencing cost, and still further aspect also needs to borrow
The auxiliary equipments such as high flux chip are helped to enter performing PCR amplification.Additionally, prior art can only could be by library label when PCR is enriched with
In adding sample, once occur sample room in experimentation mutually pollute, i.e. the contaminated sample of None- identified.
The content of the invention
It is an object of the present invention to by continuing to optimize design of primers, PCR reaction systems and PCR thermocycling programs etc. are carried
Go out a kind of compared with existing primer and method, can effectively reduce the Preference of multi-primerses so that sequencing data is more accurate, without the need for
Particular device auxiliary can enter the primer sets of performing PCR amplification enrichment target area, and complete to build using the step of the primer sets one
The method in testing sample target area nucleic acid sequencing library.
Therefore, one aspect of the present invention provides a kind of Primer composition, comprising：
First primer sets, first primer sets include the first forward primer and the first reverse primer, and described first is positive
Primer includes target area specific forward primer and the first joint；
First reverse primer includes target area specific reverse primers, reverse library label and the second joint, institute
State reverse library label to hold and the second joint between positioned at target area specific reverse primers 5 '；
Second primer sets, second primer sets include the second forward primer and the second reverse primer, and described second is positive
Primer includes the first annealing, and second reverse primer includes the second annealing；
The mutual Annealing complementary of the first annealing in first joint and second forward primer, first joint with
Product of second forward primer Jing after PCR amplifications includes the first complete sequence measuring joints；
The mutual Annealing complementary of the second annealing in second joint and second reverse primer, second joint with
Product of second reverse primer Jing after PCR amplifications includes the second complete sequence measuring joints.
Another aspect of the present invention provides above-mentioned Primer composition, the purposes in the research of immune group storehouse.
Another aspect of the present invention also provides a kind of method in structure testing sample target area nucleic acid sequencing library, including adopting
Above-mentioned primer sets are used, performing PCR amplification, to obtain amplified production, institute are entered to sample of nucleic acid of the testing sample comprising target area
State amplified production and constitute the testing sample target area nucleic acid sequencing library.
The method that another aspect of the present invention also provides a kind of determination testing sample target area nucleotide sequence, including following step
Suddenly：
Using the method in above-mentioned structure testing sample target area nucleic acid sequencing library, the target area of testing sample is built
Nucleic acid sequencing library；
The target area nucleic acid sequencing library of the testing sample is sequenced, to obtain sequencing result；
And based on the sequencing result, determine the sequence of testing sample target area nucleic acid.
Another aspect of the present invention also provides a kind of while the method for determining the target area nucleotide sequence of multiple testing samples,
Comprise the following steps：
For each in the plurality of testing sample, separately using above-mentioned structure testing sample target area
The method in nucleic acid sequencing library, builds the target area nucleic acid sequencing library of testing sample, wherein, the plurality of testing sample
Positive/negative library label is different, and the plurality of is at least 2；
The target area nucleic acid sequencing library of the plurality of testing sample is mixed, to obtain mixing library；
The mixing library is sequenced, to obtain sequencing result, the sequencing result treats test sample including the plurality of
The target area nucleic acid library sequence of product and the positive/negative library label；
And the target area nucleic acid sequencing library sequence based on the positive/negative library label to the plurality of testing sample
Make a distinction, and determine the target area nucleotide sequence of each of the plurality of testing sample.
Primer composition by the use of present invention design need not add any exogenous array as PCR binding sites, but directly
Connect using all or part of Annealing complementary area as primer of the joint of microarray dataset, improve the effective percentage of sequencing data,
So that sequencing result is more accurate, sequencing cost is reduced, reduce the difficulty of data analysiss.And by second it is positive/negative to
Primer pair product plays main enrichment, second it is positive/negative be single primer to primer, amplification preference, Neng Gouyou will not be introduced
Effect reduces the Preference of multi-primerses.The method in structure library of the invention in addition compared with the conventional method, in reverse transcription
To each sample sample library label is added, it is to avoid the mutual pollution of sample room during subsequent experimental.And without the need for single tube
Or the auxiliary PCR reactions of the particular device such as high flux chip, can complete the enrichment process of product, behaviour using common PCR equipment
Make simple, low cost, attenuating sample loss, efficiency high, cycle-index is few, take short, reduction manual operation error, sequencing result
Accurately, it is reliable, favorable repeatability.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from the description with reference to accompanying drawings below to embodiment
It is substantially and easy to understand, wherein
Fig. 1 is the schematic diagram that the first reverse primer carries out reverse transcription to mRNA in one embodiment of the invention.
Fig. 2 is the signal that the first forward primer and the second primer sets enter performing PCR to cDNA 1st in one embodiment of the invention
Figure.
Fig. 3 is that the first forward primer and the second primer sets are entered performing PCR and shown to cDNA 1st in another embodiment of the present invention
It is intended to.
Fig. 4 is the schematic flow sheet of the method that testing sample target area nucleotide sequence is determined in one embodiment of the invention.
Fig. 5 is in one embodiment of the invention while determining the stream of the method for the target area nucleotide sequence of multiple testing samples
Journey schematic diagram.
Specific embodiment
Embodiments of the invention are described below in detail.Below with reference to Description of Drawings embodiment be it is exemplary,
It is only used for explaining the present invention, and is not considered as limiting the invention.
It should be noted that term " first ", " second ", " the 3rd ", " the 4th " are only used for describing purpose, and it is not understood that
To indicate or implying relative importance or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ",
One or more this feature can be expressed or be implicitly included to " second ", " the 3rd ", the feature of " the 4th ".Further,
In describing the invention, unless otherwise stated, " multiple " are meant that two or more.
According to an aspect of the present invention, the present invention provides a kind of Primer composition, and the Primer composition is included：
First primer sets, first primer sets include the first forward primer and the first reverse primer, and described first is positive
Primer includes target area specific forward primer and the first joint；
First reverse primer includes target area specific reverse primers, reverse library label and the second joint, institute
State reverse library label to hold and the second joint between positioned at target area specific reverse primers 5 '；
Second primer sets, second primer sets include the second forward primer and the second reverse primer, and described second is positive
Primer includes the first annealing, and second reverse primer includes the second annealing；
The mutual Annealing complementary of the first annealing in first joint and second forward primer, first joint with
Product of second forward primer Jing after PCR amplifications includes the first complete sequence measuring joints；
The mutual Annealing complementary of the second annealing in second joint and second reverse primer, second joint with
Product of second reverse primer Jing after PCR amplifications includes the second complete sequence measuring joints.
Embodiments in accordance with the present invention, by the use of the present invention design Primer composition need not add any exogenous array as
PCR binding sites, but all or part of Annealing complementary area as primer in joint is directly used, improve sequencing data
Effective percentage so that sequencing result is more accurate, reduces sequencing cost, reduces the difficulty of data analysiss.And by
Two it is positive/negative play main enrichment to primer pair product, second it is positive/negative be single primer to primer, amplification will not be introduced inclined
It is good, can effectively reduce the Preference of multi-primerses.
Embodiments in accordance with the present invention, first forward primer also includes positive library label, the positive library mark
Sign and be located between the end of target area specific forward primer 5 ' and the first joint.
The library label is used to distinguish different sample libraries, can be after performing PCR amplification is entered, by the PCR of multiple samples
Product carries out mixing sequencing, and then the difference based on library label, and the samples sources of each sequence are made a distinction.Such as index/
Barcode labels, the length of the library label is 6～12bp.
Embodiments in accordance with the present invention, the positive/negative library label can be the same or different.Preferably, positive/negative text
Storehouse label is identical.
Embodiments in accordance with the present invention, first joint can be the same or different with the described first annealing, preferably
, first joint is identical with the first annealing.
Embodiments in accordance with the present invention, second joint can be the same or different with the described second annealing, preferably
, second joint is identical with the second annealing.
The length of embodiments in accordance with the present invention, first annealing and the second annealing is 16-25bp.
Embodiments in accordance with the present invention, first joint is included with the product after the second forward primer PCR amplifications
First complete sequence measuring joints.Preferably, second forward primer is the first complete sequence measuring joints.
Embodiments in accordance with the present invention, second joint is included with the product after the second reverse primer PCR amplifications
Second complete sequence measuring joints.Preferably, second reverse primer is the second complete sequence measuring joints.
A specific example of the invention, the product after first joint and the second forward primer PCR amplification
P5 sequence measuring joints of the thing comprising illumina microarray datasets, after second joint is expanded with the second reverse primer PCR
P7 sequence measuring joints of the product comprising illumina microarray datasets.Preferably, the described first complete sequence measuring joints are illumina surveys
The P5 sequence measuring joints of sequence platform.The second complete sequence measuring joints are the P7 sequence measuring joints of illumina microarray datasets.
Or first joint includes illumina microarray datasets with the product after the second forward primer PCR amplifications
P7 sequence measuring joints, the product after second joint and the second reverse primer PCR amplification is flat comprising illumina sequencings
The P5 sequence measuring joints of platform.Preferably, the described first complete sequence measuring joints are the P7 sequence measuring joints of illumina microarray datasets, described
Second complete sequence measuring joints are the P5 sequence measuring joints of illumina microarray datasets.
A specific example of the invention, the product after first joint and the second forward primer PCR amplification
P1 sequence measuring joints of the thing comprising Ion Torrent/Proton microarray datasets, second joint and second reverse primer
A sequence measuring joints of the product comprising Ion Torrent/Proton microarray datasets after PCR amplifications.Preferably, described first is complete
Sequence measuring joints are the P1 sequence measuring joints of Ion Torrent/Proton microarray datasets, and the second complete sequence measuring joints are Ion
The A sequence measuring joints of Torrent/Proton microarray datasets.
A specific example of the invention, the product after first joint and the second forward primer PCR amplification
A sequence measuring joints of the thing comprising 454 microarray datasets, the product bag after second joint and the second reverse primer PCR amplification
B sequence measuring joints containing 454 microarray datasets.Preferably, the described first complete sequence measuring joints are the A sequence measuring joints of 454 microarray datasets,
The second complete sequence measuring joints are the B sequence measuring joints of 454 microarray datasets.
A specific example of the invention, when first forward primer is not comprising positive library label, is used for
Ion Torrentt/Proton microarray datasets.
A specific example of the invention, when first forward primer is comprising positive library label, is used for
Illumina microarray datasets or 454 microarray datasets.
Embodiments in accordance with the present invention, the target area specific forward primer and the target area specificity it is reverse
The length of primer is 18-25bp.
Embodiments in accordance with the present invention, the target area specific forward primer includes B-cell receptor or φt cell receptor
V genes conserved region specific primer, the target area specific reverse primers include B-cell receptor or φt cell receptor
C genes conserved region specific primer.
According to an aspect of the present invention, the present invention provides purposes of the above-mentioned Primer composition in the research of immune group storehouse.
According to a further aspect in the invention, the present invention provides a kind of structure testing sample target area nucleic acid sequencing library
Method, including using above-mentioned primer sets, to nucleic acid of the testing sample comprising target area performing PCR amplification is entered, and is produced with obtaining amplification
Thing, the amplified production constitutes the testing sample target area nucleic acid sequencing library.
The mol ratio of embodiments in accordance with the present invention, first forward primer and the second forward primer is 1:1～1:10,
First forward primer and the second forward primer sum and the mol ratio of the second reverse primer are 1:1.
Embodiments in accordance with the present invention, the PCR reaction systems are：
Embodiments in accordance with the present invention, the PCR response procedures are：
Pcr amplification reaction is carried out using the primer sets of the present invention, it is only necessary to which 10～25 circulations can complete sample of nucleic acid
Enrichment, circulation is few, and the response time is short, efficiency high.
Embodiments in accordance with the present invention, build testing sample target area nucleic acid sequencing library method further include with
Lower step：
(1) for each target area, design is applied to the target area of the amplification target area nucleic acid amplification
Specificity is positive/negative to primer, and the first joint and the second joint, the second forward primer, the second reverse primer and positive/negative library are marked
Sign, target area specificity is positive/negative to primer, the first joint and the second joint and positive/negative library label, synthesized,
Obtain the first forward primer and the first reverse primer；
The present invention using complete sequence measuring joints it is all or part of as the first forward primer and the second forward primer, first
The Annealing complementary area of reverse primer and the second reverse primer, without the need for introducing exogenous array, it is more accurate to obtain sequencing result, reduces
Sequencing cost, reduces the difficulty of subsequent data analysis.
(2) mixing of the RNA of testing sample, the first reverse primer and cDNA synthetic agents is carried out into cDNA 1st synthesis；
Prior art needs library label could be added in sample in PCR, if sample room occur mutual for experimentation
Pollution, i.e. None- identified.And sample library label is introduced cDNA 1st by the present invention in reverse transcription by reverse transcription, while handle
The binding site of the second reverse primer is introduced in cDNA 1st, as long as can using the second reverse primer in follow-up PCR reactions
To carry out amplified reaction.Namely the present invention in reverse transcription will each sample added sample library label, it is to avoid follow-up
The mutual pollution of experimentation sample room.
Preferably, performing PCR reaction is entered after the mixing in PCR pipe.
Preferably, the testing sample RNA is messenger RNA (mRNA).
Preferably, the cDNA synthetic agents include that dideoxyribonucleotide triphosphate mixed liquor (dNTP mi), 5x1 chains delay
Rush liquid (5 × 1st strand buffer), dithiothreitol, DTT (DTT), RNase inhibitor (RNAseOUT), reverse transcription III
(SuperScriptTMIII), RNase mixture (RNase mix).
Preferably, the synthesis step includes：First testing gene group RNA, the first reverse primer and dNTP mix are mixed
Close, carry out RNA degeneration；After degeneration terminates, add 5 × 1st strand buffer, DTT, RNAseOUT,
SuperScriptTMIII, enters performing PCR reaction, and response procedures are 50 DEG C of 50min, 70 DEG C of 15min；After PCR reactions terminate, add
RNase mix, 37 DEG C of incubation 30min, obtain cDNA 1st.
(3) the cDNA 1st that synthesis is obtained are mixed into the first forward primer, the second primer sets and PCR reaction reagents
Performing PCR is expanded, and the mol ratio of first forward primer and the second forward primer is 1:1～1:10, first forward primer
It is 1 with the molal quantity of the second forward primer sum and the second reverse primer:1.
Preferably, performing PCR reaction is entered after the mixing in PCR pipe.
Preferably, the PCR response procedures are：
(4) after PCR reactions terminate, PCR primer is reclaimed, magnetic beads for purifying is carried out, according to 0.8～1 times of body of PCR primer volume
Product adds magnetic bead, purified product to be nucleic acid library.
Preferably, the magnetic beads for purifying carries out purification using AMPure XP DNA purification kits.
The method in the structure testing sample target area nucleic acid sequencing library that the present invention is provided, without the need for extra sequence measuring joints
The step of connection, connection product purification, without the auxiliary PCR reactions of the particular device such as single tube or high flux chip, using common
PCR equipment can complete the enrichment process of product, it is simple to operate, low cost, lower sample loss, efficiency high, cycle-index
It is few, short, reduction manual operation error is taken, sequencing result is accurate, reliability, favorable repeatability.
Some specific examples of the invention, the side in the structure testing sample target area nucleic acid sequencing library of the present invention
Method can also be comprised the following steps：
The first primer sets that synthesis is obtained are carried out into pre- PCR amplifications and detected through gel electrophoresis, to verify the first primer sets
Whether meet the requirements, wherein so that whether the single band for meeting designed size can be amplified as criterion.
Some specific examples of the invention, the method for building testing sample target area nucleic acid sequencing library is further
Comprise the following steps：
The nucleic acid library for building is carried out into Library Quality detection, it is possible to use such as Agilent2100Bioanalyzer
Or Caliper Bioanalyzer, or ABI StepOnerPlus Real-Time PCR System carry out mass concentration, piece
The detection of section size distribution and molar concentration.After qualified after testing, you can upper machine sequencing.
According to a further aspect in the invention, the present invention also provides a kind of side of determination testing sample target area nucleotide sequence
Method.
Embodiments in accordance with the present invention, with reference to Fig. 4, the method is comprised the following steps：
S101：Build the target area nucleic acid sequencing library of testing sample
According to the method in above-mentioned structure testing sample target area nucleic acid sequencing library, the target area of testing sample is built
Nucleic acid sequencing library.
S102：Target area nucleic acid sequencing library is sequenced
The target area nucleic acid sequencing library of the testing sample is sequenced, to obtain sequencing result.
Some embodiments of the invention, product after first joint is expanded with the second forward primer PCR
P5 sequence measuring joints of the thing comprising illumina microarray datasets, after second joint is expanded with the second reverse primer PCR
During P7 sequence measuring joints of the product comprising illumina microarray datasets, using Illumina microarray datasets the sequencing is carried out.
Or product after first joint is expanded with the second forward primer PCR is flat comprising illumina sequencings
The P7 sequence measuring joints of platform, second joint is sequenced with the product after the second reverse primer PCR amplifications comprising illumina
During the P5 sequence measuring joints of platform, using Illumina microarray datasets the sequencing is carried out.
Other embodiments of the invention, after first joint is expanded with the second forward primer PCR
P1 sequence measuring joints of the product comprising Ion Torrent/Proton microarray datasets, second joint and second reverse primer
When product after PCR amplifications includes the A sequence measuring joints of Ion Torrent/Proton microarray datasets, using Ion Torrent/
Proton microarray datasets carry out the sequencing.
Other embodiments of the invention, after first joint is expanded with the second forward primer PCR
A sequence measuring joints of the product comprising 454 microarray datasets, the product after second joint and the second reverse primer PCR amplification
During the B sequence measuring joints comprising 454 microarray datasets, using 454 microarray datasets the sequencing is carried out.
S103：Determine testing sample target area nucleotide sequence
Based on the sequencing result, the sequence of testing sample target area nucleic acid is determined.
In accordance with a further aspect of the present invention, the present invention also provides a kind of while determining the target area core of multiple testing samples
The method of acid sequence.
Embodiments in accordance with the present invention, with reference to Fig. 5, the method is comprised the following steps：
S201：Separately build each the target area nucleic acid sequencing library in multiple testing samples
For each in the plurality of testing sample, separately according to above-mentioned structure testing sample target area
The method in nucleic acid sequencing library, builds the target area nucleic acid sequencing library of testing sample, wherein, the plurality of testing sample
Positive/negative library label is mutually different, and the plurality of is at least 2.
S202：The target area nucleic acid sequencing library of multiple testing samples is mixed
The target area nucleic acid sequencing library of multiple testing samples is mixed, to obtain mixing library.
S203：It is sequenced to mixing library
The mixing library is sequenced, to obtain sequencing result, the sequencing result includes the plurality of to be measured
The target area nucleic acid library sequence of sample and the positive/negative library label.
Some embodiments of the invention, product after first joint is expanded with the second forward primer PCR
P5 sequence measuring joints of the thing comprising illumina microarray datasets, after second joint is expanded with the second reverse primer PCR
During P7 sequence measuring joints of the product comprising illumina microarray datasets, using Illumina microarray datasets the sequencing is carried out.
Or product after first joint is expanded with the second forward primer PCR is flat comprising illumina sequencings
The P7 sequence measuring joints of platform, second joint is sequenced with the product after the second reverse primer PCR amplifications comprising illumina
During the P5 sequence measuring joints of platform, using Illumina microarray datasets the sequencing is carried out.
Other embodiments of the invention, after first joint is expanded with the second forward primer PCR
P 1 sequence measuring joints of the product comprising Ion Torrent/Proton microarray datasets, second joint reversely draws with described second
When product after thing PCR amplifications includes the A sequence measuring joints of Ion Torrent/Proton microarray datasets, using Ion Torrent/
Proton microarray datasets carry out the sequencing.
Other embodiments of the invention, after first joint is expanded with the second forward primer PCR
A sequence measuring joints of the product comprising 454 microarray datasets, the product after second joint and the second reverse primer PCR amplification
B sequence measuring joints comprising 454 microarray datasets, using 454 microarray datasets the sequencing is carried out.
S204：Determine the target area nucleotide sequence of each in multiple testing samples
The sequence in the target area nucleic acid sequencing library of the plurality of testing sample is entered based on the positive/negative library label
Row is distinguished, and determines the target area nucleotide sequence of each of the plurality of testing sample.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that showing below
Example is only used for explaining the present invention, and is not considered as limiting the invention.Except as otherwise explaining, it is related to not in following examples
Reagent, sequence (joint, label and primer), software and the instrument especially explained all is conventional commercial product or is increased income.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Embodiment one
Design primer
With the reference sequences of IMGT data bases as foundation, the specific primer of BCR, TCR is designed.First reverse primer is in C
The conserved regions design of gene, while first reverse primer connects reverse library label at the 5' ends of C gene specific primers, instead
Connect the second joint to the 5' ends of library label, see the first reverse primer in Fig. 1.First forward primer includes V genes conserved region
Specific primer and the first joint, see the first forward primer in Fig. 2, or the specific primer including V genes conserved region, just
To library label and the first joint, the first forward primer in Fig. 3 is seen.Positive/negative library label is used to distinguish different samples.The
Two forward primers include the first annealing, and as shown in Figure 2, the second reverse primer includes the second annealing, as shown in Figure 3.
When entering performing PCR and reacting, what is mainly worked is the joint at upstream and downstream two ends, that is, the second forward primer and the
Two reverse primers.The present invention does not introduce one section of sequence of external source as PCR binding sites, and is the use of sequence measuring joints
Partial sequence, i.e., the 3' ends 16-25bp of complete sequence measuring joints reversely draws as the first forward primer and the second forward primer, first
Thing and the second reverse primer Annealing complementary area, cause this method to carry out end and repair just because of this special design
It is multiple, plus ' A ' and the first-class process of adjunction, directly carry out purpose product and be enriched with while completing library construction.It is exemplified below, designs 2
Sample primer sets sequence.
12 groups of primer sets sequences of table
It is prepared by sample library
1st, density gradient separation human peripheral blood single nucleus cell
Taken a blood sample (anticoagulant typically has edta edta) using the sterile blood sampling pipe containing anticoagulant, using pouring
Bar cell separation liquid, such as Ficoll-Paque PLUS lymphocyte separation mediums or Percoll lymphocyte separation mediums carry out close
Degree gradient separations PBMC.
2nd, RNA is extracted
Total serum IgE is extracted using Trizol regeant methods or extracts kit etc..
3rd, DNA digestion
The DNA remained in RNA samples is removed using DNA digestive enzyme.
4th, cDNA 1st synthesis
Reverse transcription is carried out to RNA using reverse transcription and reverse transcription primer, the chains of cDNA 1 are obtained, and adds RNA digestive enzyme,
Digestion RNA.As shown in figure 1, the first reverse primer carries out reverse transcription to mRNA.
4.1 prepare (1 sample) in 0.2ml PCR pipes by following system.
| Component | Volume (μ L) |
| The RNA (20ng/ul) obtained after DNA has been digested | 10 |
| First reverse primer (2pmol/ul) | 1 |
| dNTP mix(10mM) | 1 |
| Jing pyrocarbonic acid diethyl esters process water (DEPC water) | 1 |
4.2 by mixture be placed in 65 DEG C 5 minutes so that RNA degeneration, degeneration terminates, and places 1min on ice, after brief centrifugation,
0.2ml PCR pipes during following mixed solution is added to into 4.1.
| Component | Volume μ L |
| 5x1 chain buffer (5 × 1st strand buffer) | 4 |
| 100mM M dithiothreitol, DTTs (DTT) | 1 |
| RNase inhibitor (RNAseOUT) (40U/ μ L) | 1 |
| Reverse transcription III (SuperScriptTM III) (200U/ μ L) | 1 |
Wherein, the cumulative volume of PCR pipe is 20 μ L.Slight concussion is mixed.
4.3 react in PCR instrument
50℃50min。
70℃15min
4.4 add 1 μ LRNase mix, slight concussion simultaneously thoroughly to mix, 37 DEG C of incubation 30min.
4.5 to specifications, using AMpure XP purification cDNA1 chain samples, with 18ul nuclease free Water Sproading cDNA1
Chain sample.
After this step terminates, reactant mixture can be stored in -20 DEG C.
5th, the PCR conditions of particular design are enriched with target area and complete library construction
The primer that PCR is used includes the first forward primer (V areas forward primer) and the second primer sets, wherein the first forward direction is drawn
The mol ratio of thing and the second forward primer is 1:1～1:10, first forward primer and the second forward primer sum and second
The mol ratio of reverse primer is 1:1.After this step PCR terminates, complete sequence measuring joints, library construction knot are contained in purpose fragment two ends
Beam.See Fig. 2 and Fig. 3, the first forward primer and the second primer sets enter performing PCR to cDNA 1st.
The 5.1 cDNA 1st for obtaining synthesis are enriched with as template PCR.Following mixed solution is existed
PCR reaction systems are prepared in the PCR pipe of 200 μ L.
PCR reaction conditions are：
6th, sequencing library purification
The sequencing library of structure can carry out purification, respectively magnetic beads for purifying, purification column purification and agarose with three kinds of methods
Gel electrophoresis recovery purifying.
After PCR reactions terminate, PCR primer is transferred in 1 1.5mL centrifuge tube, with AMPure XP DNA purified reagent
Sample after the amplification of box (SPRI beads) purification.
7th, library detection
Agilent 2100Bioanalyzer analysis system detect library inserts size and content；Q-
The concentration in PCR accurate quantifications library.
High-flux sequence
The sequencing library of structure is sequenced on high-flux sequence platform, high-flux sequence platform includes Illumina
The Ion Torrent sequencings of Hiseq and Miseq microarray datasets, the microarray datasets of Roche 454 and Life Technologies are flat
Platform etc..
Sequencing data is analyzed
The data that sequencing is obtained are carried out into mass filter and length filtration removes depollution and joint sequence；Statistical result bag
Include：Sequence (Reads) length of measure, data throughput.Then the sequence for obtaining is compared, according to V on IMGT,
Position is found out and determined to V, (D) J genes by the definition of (D) J genes, and count V, the multiformity of (D) J genes.
CDR3 is found out according to the definition on IMGT, and counts the length and multiformity of CDR3.
In the description of this specification, the description of reference term " one embodiment ", " specific example " etc. means to combine is somebody's turn to do
Embodiment or the specific features of example description, structure, material or feature are contained at least one embodiment of the present invention or show
In example.In this manual, identical embodiment or example are not necessarily referring to the schematic representation of above-mentioned term.And,
The specific features of description, structure, material or feature can be in any one or more embodiments or example with suitable
Mode is combined.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
Enclose and be defined, on the premise of without departing from design spirit of the present invention, this area ordinary skill technical staff is to the technology of the present invention
Various modifications and improvement that scheme is made, all should fall in the protection domain of claims of the present invention determination.
SEQUENCE LISTING
<110>Guangzhou company limited of medical test institute of essence section
<120>Primer composition, its purposes, the method for building library and determination nucleotide sequence
<130> CN83656
<160> 22
<170> PatentIn version 3.5
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<211> 43
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<213> Artificial
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<223>First forward primer
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Claims (14)
1. a kind of Primer composition, it is characterised in that include：
First primer sets, first primer sets include the first forward primer and the first reverse primer, first forward primer
Comprising target area specific forward primer and the first joint；
First reverse primer includes target area specific reverse primers, reverse library label and the second joint, described anti-
Target area specific reverse primers 5 ' are located to library label to hold and the second joint between；
Second primer sets, second primer sets include the second forward primer and the second reverse primer, second forward primer
Comprising the first annealing, second reverse primer includes the second annealing；
The mutual Annealing complementary of the first annealing in first joint and second forward primer, first joint with it is described
Product of second forward primer Jing after PCR amplifications includes the first complete sequence measuring joints；
The mutual Annealing complementary of the second annealing in second joint and second reverse primer, second joint with it is described
Product after second reverse primer PCR amplifications includes the second complete sequence measuring joints.
2. Primer composition as claimed in claim 1, it is characterised in that first forward primer also includes positive library mark
Sign, the positive library label is located at target area specific forward primer 5 ' and holds and the first joint between.
3. Primer composition as claimed in claim 1, it is characterised in that the length of first annealing and the second annealing is
16-25bp。
4. Primer composition as claimed in claim 1, it is characterised in that first joint and the second forward primer Jing
P5 sequence measuring joints of the product comprising illumina microarray datasets after PCR amplifications, second joint reversely draws with described second
Product P7 sequence measuring joints comprising illumina microarray dataset of the thing Jing after PCR amplifications；
Or product of first joint with second forward primer Jing after PCR amplifications includes illumina microarray datasets
P7 sequence measuring joints, product of second joint with second reverse primer Jing after PCR amplifications is flat comprising illumina sequencings
The P5 sequence measuring joints of platform；
Or product of first joint with second forward primer Jing after PCR amplifications includes Ion Torrent/Proton
The P1 sequence measuring joints of microarray dataset, product of second joint with second reverse primer Jing after PCR amplifications includes Ion
The A sequence measuring joints of Torrent/Proton microarray datasets；
Or product A comprising 454 microarray datasets of first joint with second forward primer Jing after PCR amplifications is sequenced
Joint, product B comprising 454 microarray datasets sequencing of second joint with second reverse primer Jing after PCR amplifications connects
Head.
5. Primer composition as claimed in claim 1, it is characterised in that when the first forward primer is not comprising positive library label
When, for Ion Torrentt/Proton microarray datasets.
6. Primer composition as claimed in claim 1, it is characterised in that when first forward primer is comprising positive library mark
During label, for illumina microarray datasets or 454 microarray datasets.
7. Primer composition as claimed in claim 1, it is characterised in that the target area specific forward primer includes B
The specific primer of the V genes conserved region of cell receptor or φt cell receptor, the target area specific reverse primers include B
The specific primer of the C genes conserved region of cell receptor or φt cell receptor.
8. the Primer composition described in claim 1 immune group storehouse research in purposes.
9. it is a kind of build testing sample target area nucleic acid sequencing library method, it is characterised in that using claim 1～7
Primer sets described in any one, to nucleic acid of the testing sample comprising target area performing PCR amplification, to obtain amplified production, institute are entered
State amplified production and constitute the testing sample target area nucleic acid sequencing library.
10. method according to claim 9, it is characterised in that first forward primer and second forward primer
Mol ratio be 1:1～1:10, first forward primer and the second forward primer sum and second reverse primer
Mol ratio be 1:1.
11. methods according to claim 9, it is characterised in that the response procedures of PCR amplification are：
12. methods according to claim 9, it is characterised in that further comprising the steps：
(1) for each target area, design is special suitable for the target area of the amplification target area nucleic acid amplification
Property it is positive/negative to primer, it is the first joint and the second joint, second positive/negative to primer and positive/negative library label, target area is special
The opposite sex is positive/negative to primer, the first joint and the second joint and positive/negative library label, is synthesized, and obtains the first forward primer
With the first reverse primer；
(2) testing gene group RNA, the first reverse primer and cDNA synthetic agents are mixed, to carry out cDNA 1st synthesis；
(3) mixing the cDNA 1st that synthesis is obtained with the first forward primer, the second primer sets and PCR reaction reagents is carried out
PCR is expanded, and the mol ratio of first forward primer and the second forward primer is 1:1～1:10, first forward primer with
Second forward primer sum is 1 with the molal quantity of the second reverse primer:1；
(4) after PCR reactions terminate, PCR primer is reclaimed, carries out magnetic beads for purifying, added according to 0.8～1 times of volume of PCR primer volume
Enter magnetic bead, purified product is nucleic acid library.
A kind of 13. methods for determining testing sample target area nucleotide sequence, it is characterised in that comprise the following steps：
Method according to any one of claim 9～12, builds the target area nucleic acid sequencing library of testing sample；
The target area nucleic acid sequencing library of the testing sample is sequenced, to obtain sequencing result；
And based on the sequencing result, determine the sequence of testing sample target area nucleic acid.
14. is a kind of while the method for determining the target area nucleotide sequence of multiple testing samples, it is characterised in that including following step
Suddenly：
For each in the plurality of testing sample, the separately side according to any one of claim 9～12
Method, builds the target area nucleic acid sequencing library of testing sample, wherein, the positive/negative library label of the plurality of testing sample is mutual
Differ, the plurality of is at least 2；
The target area nucleic acid sequencing library of the plurality of testing sample is mixed, to obtain mixing library；
The mixing library is sequenced, to obtain sequencing result, the sequencing result includes the plurality of testing sample
Target area nucleic acid library sequence and the positive/negative library label；
And the target area nucleic acid sequencing library sequence of the plurality of testing sample is carried out based on the positive/negative library label
Distinguish, and determine the target area nucleotide sequence of each of the plurality of testing sample.
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/CN2017/100426 WO2018095108A1 (en) | 2016-09-27 | 2017-09-04 | Primer composition, use thereof, and methods for constructing library and for determining nucleic acid sequence |
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| CN201610862251X | 2016-09-27 | ||
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| WO2018095108A1 (en) * | 2016-09-27 | 2018-05-31 | 广州精科医学检验所有限公司 | Primer composition, use thereof, and methods for constructing library and for determining nucleic acid sequence |
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| CN106048009B (en) * | 2016-06-03 | 2020-02-18 | 人和未来生物科技（长沙）有限公司 | Label joint for ultralow frequency gene mutation detection and application thereof |
| CN106636063A (en) * | 2016-09-27 | 2017-05-10 | 广州精科医学检验所有限公司 | Primer compound, application thereof and method for establishing library and confirming nucleotide sequence |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018095108A1 (en) * | 2016-09-27 | 2018-05-31 | 广州精科医学检验所有限公司 | Primer composition, use thereof, and methods for constructing library and for determining nucleic acid sequence |
| CN107365867A (en) * | 2017-09-04 | 2017-11-21 | 天津华大医学检验所有限公司 | A kind of Primer composition and its application for being used to detect genome target region |
| CN116064753A (en) * | 2018-08-28 | 2023-05-05 | 深圳联合医学科技有限公司 | Method for constructing high-efficiency sequencing library, primer set and kit |
| CN113166801A (en) * | 2018-12-20 | 2021-07-23 | 深圳华大智造科技股份有限公司 | PCR primer, PCR amplification method and application |
| CN111349718A (en) * | 2018-12-21 | 2020-06-30 | 深圳华大智造科技有限公司 | Primer set for pathogen nucleic acid amplification, pathogen nucleic acid detection library construction method and pathogen detection method |
| CN111662969A (en) * | 2020-05-18 | 2020-09-15 | 北京优吉科技有限公司 | Gene transcription region multi-variable region sequencing method |
| CN112301099A (en) * | 2020-11-30 | 2021-02-02 | 南方科技大学 | Primer group for amplifying B lymphocyte immune repertoire and application thereof |
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| WO2018095108A1 (en) | 2018-05-31 |
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