CN106754904B - The specific molecular label of cDNA a kind of and its application - Google Patents
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Abstract
The specific molecular label and its application, the specific molecular label of a kind of cDNA includes: the universal primer sequence as shown in SEQ ID NO.1；The transposase joint sequence P7 as shown in SEQ ID NO.2；Random sequence: (N)n, N is random sequence, total n；With poly (T) sequence for sharing 30 T alkali, specific nucleotide sequence such as SEQ ID NO.3 institute.Specific molecular label is applied to cDNA to build in library, during RNA reverse transcription is at full-length cDNA, the specific molecular label is added in the end 5` of each cDNA, make each gene be it is unique, subsequent information analysis can according to the sequence of the special molecular label quantify sample in each gene.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to the specific molecular label of cDNA a kind of and its application.
Background technique
In past twenty or thirty year, the extensive gene sequence analysis of two generations sequencing has greatly pushed genome and has turned
The sequence of record group is analyzed.The sequencing of transcript profile is not because needing to be pre-designed entire transcript profile probe and can detecte unknown
Transcript has gradually replaced the transcriptome analysis scheme that traditional chip gene expression profile becomes preferred.
Transcript profile refers in a certain moment, by the whole RNA of cell transcription out, including mRNA and non-coding RNA.
Transcription group is exactly from the expression of rna level research gene, the case where studying genetic transcription in cell in integral level
And the rule of transcriptional control.Different from genome, transcription group research contains the restriction in time and space, and same cell is not
Under same growth period and growing environment, expression conditions are not exactly the same.Traditional transcriptome analysis needs to count
Ten thousand are analyzed to hundreds of thousands of a cells, this be averaged a large amount of gene expression based on the method for population level, can not
Disclose the heterogeneity of gene expression between cell.In addition, when target cell number is rare and is difficult to cultivate, this traditional point
Analysis method is with regard to helpless, it is necessary to use unicellular transcriptome analysis technology.
The resolution ratio of unicellular transcript profile sequencing technologies is accurate to individual cells, to distinguish various cells in heterogeneous population
The transcript profile feature of type provides strong tool.Traditional banking process uses the methods of ultrasonic wave, atomization fragmentation
DNA needs larger amount of starting material, then by end repair, addition connector and etc. realization.However based on single celled
Transcript profile sequencing starting total serum IgE amount is few, and this method is not suitable for, this is that many potential users is allowed to bring down a bit.
Construction cDNA library is the key technology step of unicellular transcriptome analysis, and method includes that separation is unicellular, thin
Cellular lysate, mRNA reverse transcription are at cDNA, cDNA fragmentation and the several steps of adjunction head, amplified library.Single celled transcript profile is surveyed
Sequence starting total serum IgE amount is few, uses poly thymidine (oligo-dT) as reverse in the case where not removing rRNA
It records primer and obtains a large amount of full-length cDNA, using template switch (template-switching) activity of reverse transcriptase in cDNA
3 ends ` add the preceding paragraph joint sequence, this section of joint sequence can be used for full-length cDNA amplification and then obtain the sequence of entire mRNA,
To avoid the Preference at 3 ends `.It can be added while interrupting at the both ends fragmentation DNA using the banking process of transposase
Connector, by cumbersome DNA fragmentation, end repair and connection reaction and etc. become the simple enzymatic reaction of a step, it is this
Method greatly reduces starting template amount and sample processing time.
The research of transcript profile can study gene function and gene structure from integral level, disclose point of bioprocess
Handset reason, quantify each transcript wherein expression variation, be therefore widely used in biological study, medicament research and development,
The fields such as basic research and clinical diagnosis.However, determining the absolute number of different genes in two different plant species or simple sample
The very challenging property of abundance is measured, due to the Preference of PCR amplification, the mRNA of variable expression is in the PCR amplification for continuing library after
The information reflected afterwards has inaccuracy, and subsequent analysis of biological information data can not true response gene expression quantity.
Summary of the invention
The purpose of the invention is to provide the specific molecular label of cDNA a kind of and its application, the specific molecular marks
Label are added in 5 ends ` of each cDNA, make each gene be it is unique, subsequent information analysis can be according to the spy
Each gene in the sequence quantization sample of opposite molecule label.
In order to achieve the above objectives, technical scheme is as follows:
A kind of specific molecular label of cDNA includes: universal primer sequence in full length sequence；Transposase joint sequence
P7；Random sequence: (N)n, N is random sequence, total n；With poly (T) sequence；Wherein, the universal primer sequence such as SEQ
Shown in ID NO.1, for transposase joint sequence as shown in SEQ ID NO.2, poly (T) sequence shares 30 i.e. T30VN of T base.
Preferably, the specific molecular label of cDNA of the present invention, nucleotide sequence as shown in SEQ ID NO.3,
Particular sequence is as follows: AAGCAGTGGTATCAACGCAGAGTACGTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG (N)nT30VN, wherein N is random sequence, and total n, N is selected from A, T, C, G；Preferably, n 5-10.
The specific molecular label of cDNA of the present invention builds the application in library in cDNA.The specific molecular of the cDNA
Label is also applied to build in library using the cDNA that transposase interrupts.
The present invention also provides a kind of cDNA banking process interrupted using transposase comprising following steps:
1) separation is unicellular, will discharge total serum IgE after slender cellular lysate；Using mRNA as template, spy as claimed in claim 1 or 2
Opposite molecule label is that primer reverse transcription synthesizes the first chain cDNA, and the specific molecular label is made to be added in the 5 ' of the first chain cDNA
End, and in process of reverse-transcription, using the template switch activity of reverse transcriptase in the 3 ' ends of the first chain cDNA plus the preceding paragraph connector
Sequence, the joint sequence with as universal primer sequence shown in SEQ ID NO.1 it is identical；Using the first chain cDNA as template, with such as
Universal primer sequence shown in SEQ ID NO.1 is the full-length cDNA that primer passes through PCR amplification anamorphic zone specific molecular label,
And pcr amplification product is purified；
2) full-length cDNA obtained by step 1) and transposase embedding compound are incubated for, complete cDNA fragmentation and adjunction
Head, wherein transposase embedding compound includes transposase and annealing connector；
3) PCR amplification is carried out to the fragmentation products that step 2) obtains using upstream primer N7 and downstream primer N5；Wherein,
The sequence of upstream primer N7 is as shown in SEQ ID NO.4: CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG, under
The sequence of primer N5 is swum as shown in SEQ ID NO.5: AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC；
4) pcr amplification product obtained by step 3) is purified, obtains the end the 5` text of the cDNA with specific molecular label
Library.
Further, in step 2), each method processed of the transposase embedding compound includes: preparation system: prepare system:
Transposase (10U) 12-15 μ L, annealing 5 μ L of connector, buffer 12-15 μ L is embedded, 33 μ L of total volume is gently blown and beaten with pipettor
It mixes, the reaction system prepared is placed in PCR instrument and is reacted 1.5 hours for 30 DEG C, -20 DEG C of preservations.
Transposase in the transposase embedding compound is Tn5 family transposase, wild type Tn5 transposase, saltant type
Tn5 transposase is suitable for the present invention；It is described annealing connector nucleotide sequence as shown in SEQ ID NO.6, specifically:
CTCTTATACACATCTGACGCTGCCGACGA。
Again, in step 2), the cDNA fragmentation and plus jointing method include: preparation reaction mixture: the swivel base
Enzyme embeds compound 3-7 μ l, swivel base enzyme reaction buffer solution 4-8 μ l；By the end 5` plus the specific molecular label cDNA with
Reaction mixture mixing, is blown and beaten with pipettor and is mixed；55 DEG C of reaction 10-15min in PCR instrument.
Specific molecular label of the present invention shares 4nKind, by 30 T bases of poly thereon (T) sequence with
Poly (A) tail Annealing complementary on mRNA carries out reverse transcription under the action of reverse transcriptase and obtains the first chain cDNA, specificity point
Subtab is added in the 5 ' ends of the first chain cDNA, adds one at the 3 ' ends of the first chain cDNA using the template switch activity of reverse transcriptase
Section joint sequence identical with universal primer sequence；It is obtained by such as the amplifiable synthesis of universal primer shown in SEQ ID NO.1
Full-length cDNA with specific molecular label, the full-length cDNA is without 3` and 5` Preference.
The banking process of the present invention interrupted using transposase, in cDNA fragmentation process, fragmentation fracture
Both ends are coupled with the annealing connector of transposase, i.e., in addition to the end 3` and 5` of cDNA, there is identical move back at the both ends of the DNA of fragmentation
Fiery joint sequence P5 (as shown in SEQ ID NO.6)；And transposase connector sequence is had on the 5` end-specificity molecular label of cDNA
P7 is arranged, therefore using the N7 primer with transposase joint sequence (as shown in SEQ ID NO.2) and with annealing joint sequence
The N5 primer of (as shown in SEQID NO.6) can react the end 5` of enrichment cDNA by PCR.Subsequent analysis of biological information
It can be by marking the random sequence in the specific molecular label on the end 5` of each cDNA each in library to distinguish
The absolute quantitation of cDNA is realized in the source of a cDNA.
In full-length cDNA preparation process, specific molecular label of the present invention is added in the 5 ' ends of the first chain cDNA, from
And stamped a random molecular label to each mRNA, make each gene be it is unique, subsequent
Even if having different degrees of Preference in the enrichment process of the end cDNA 5`, but for the cDNA of identical molecular label, it can be denoted as and
Derived from the same copy, therefore can distinguishing different by the different of molecular label when biological data is analyzed
MRNA, to be carried out to the copy number of mRNA accurately quantitative.
Beneficial effects of the present invention:
(1) it is built in library compared with common sequence with existing cDNA, the molecular label that the present invention designs has 4 different sequences
Part forms, this 4 parts play different effect: universal primer sequence respectively: due to reverse transcriptase in process of reverse-transcription
Template switch activity adds the preceding paragraph sequence identical with universal primer at the end 3` of cDNA, synthesizes by the way that universal primer is amplifiable
To the full-length cDNA with specific molecular label；Transposase joint sequence P7: on the 5` end-specificity molecular label of full-length cDNA
With transposase joint sequence P7, and in cDNA fragmentation process, the both ends of the DNA of fragmentation have identical annealing to connect
Header sequence P5 is connect using the upstream primer N7 with transposase joint sequence P7 (as shown in SEQ ID NO.2) and with annealing
The downstream primer N5 of header sequence P5 (as shown in SEQ ID NO.6) can react the end 5` for being only enriched with cDNA by PCR；At random
Sequence: each mRNA molecule is marked；Poly (T) sequence: the end 3` poly (A) sequence complementary pairing with mRNA, in reverse transcription
Reverse transcription synthesizes the first chain cDNA under the action of enzyme.
(2) compared with existing transposase interrupts banking process, cDNA of the invention builds the embedding of transposase used in library
Joint sequence in compound only has a kind of annealing joint sequence P5, by the upstream primer N7 for having transposase joint sequence P7
It is annealing joint sequence P5 that both ends, which can be abandoned, all during PCR with the downstream primer N5 with annealing joint sequence P5
DNA fragmentation and the end 5` for being only enriched with cDNA.Subsequent information analysis only needs the sequence at the end 5` of cDNA just to can know that its representative
MRNA, without obtaining the cDNA sequence information of overall length.
(3) molecular label provided by the present invention is added in the end 5` of each cDNA, each gene is made to have an only nothing
Two molecular label, subsequent information analysis can quantify each gene in sample according to the sequence of label, and accuracy is significantly
It improves.
(4) when the specific molecular label designed by the present invention is applied to construction cDNA library, required starting total amount RNA is low
(1-1000 cell, 10pg-10ng), individual cells can be used as starting template and carry out efficient amplification, solve rare sample
Measure insufficient problem；Greatly reduce the processing of RNA sample simultaneously, so as to avoid the loss of sample, improve operation at
Power.
Specific embodiment
Technical solution of the present invention is described in further detail below in conjunction with specific embodiment.
The specific molecular label of the design of embodiment 1 cDNA
The specific molecular label of cDNA includes:
A, universal primer sequence is used for subsequent PCR amplification full-length cDNA as shown in SEQ ID NO.1, and universal primer is specific
Sequence is as follows: AAGCAGTGGTATCAACGCAGAGTAC；
B, transposase joint sequence P7, as shown in SEQ ID NO.2, particular sequence is as follows: GTCTCGTGGGCTCGG
AGATGTGTATAAGAGACAG；
C, random sequence (N)n, wherein N is random sequence, total n；
D, poly (T) sequence, totally 30 T base, that is, T30VN；
CDNA molecular label is synthesized by method for synthesizing gene, nucleotide sequence is as shown in SEQ ID NO.3:
AAGCAGTGGTATCAACGCAGAGTAC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(N)nT30VN, wherein N
For random sequence, total n, N is selected from A, T, C, G；Preferably, n 5-10.
Embodiment 2 prepares the full-length cDNA with the specific molecular label
293 cells for selecting people are experimental material, and the Discover-sc WTA Kit of (Vazyme) is only praised using Nanjing promise
V2 (N711) kit carries out transcript profile amplification.Used reagent and material is provided by the kit, for example, unicellular split
Solve liquid, RNase Inhibitor, RT Buffer, DTT, TSOligo, reverse transcriptase, high fidelity enzyme etc..
(1) preparation of samples
1) 10 × Sample Buffer is prepared in the following order, is mixed gently with pipettor, and of short duration is collected by centrifugation.
Table 1
| Component | Volume |
| Unicellular lysate | 19μl |
| RNase Inhibitor | 1μl |
| Total volume | 20μl |
2) in the PCR pipe for taking 10 × Sample Buffer to 0.2ml of 1 μ l, the nuclease free pure water of 4 μ l is added
(Nuclease-free H2O)。
3) single celled separation
293 cell suspending liquids for taking fresh cultured, are collected by centrifugation at normal temperature, abandon culture solution and PBS is added to be resuspended, aobvious being inverted
Single celled selection separation is carried out under micro mirror, and by the isolated unicellular step 2) that is added to containing 10 × Sample
In the PCR pipe of the 0.2ml of Buffer, tube wall mixing is flicked, 5min is incubated at room temperature.
(2) first chain cDNA synthesis
1) sample after cracking is placed on ice, prepares reaction mixture according to table 2, then 4 μ l reaction mixtures are added
In the unicellular sample cracked to 6 μ l, mixed gently with pipettor, of short duration be collected by centrifugation is placed on ice.
Table 2
| Component | Volume |
| The special molecular label as shown in SEQ ID NO.3 | 2μl |
| dNTP | 2μl |
| Total volume | 4μl |
2) 72 DEG C of reaction 3min in PCR instrument, after be immediately placed on 2min on ice.
3) by PCR instrument be preheated to 42 DEG C it is spare.
4) reaction mixture is prepared according to table 3, is mixed gently with pipettor, of short duration be collected by centrifugation is placed on ice.
Table 3
| Component | Volume |
| Nuclease-free H2O | 2.5μl |
| Step 2) reaction product | 10μl |
| RT Buffer | 4μl |
| DTT | 1μl |
| RNase Inhibitor | 0.5μl |
| TS Oligo | 1μl |
| Reverse transcriptase | 1μl |
| Total volume | 20μl |
5) PCR reaction as shown in table 4 is carried out, after reaction place the product on ice, product is the first chain cDNA synthesis
Product.
Table 4
| Heat lid | 105℃ |
| 42℃ | 90min |
| 70℃ | 15min |
| 4℃ | It maintains |
This step reverse transcription has synthesized the first chain cDNA, and identical as universal primer sequence at the 3 ' ends of cDNA plus the preceding paragraph
Joint sequence.
(3) with the full-length cDNA amplification of molecular label
It is primer by PCR amplification synthesis full-length cDNA by the universal primer sequence as shown in SEQ ID NO.1, specifically
Steps are as follows:
1) PCR reaction solution as shown in table 5 is configured, is mixed gently with pipettor, of short duration be collected by centrifugation is placed on ice.
Table 5
2) program as shown in table 6 is run in PCR instrument:
Table 6
(4) PCR product purifies
PCR product is adsorbed using the VAHTS DNA Clean Beads (N411) that (Vazyme) is only praised in Nanjing promise, 80%
Ethyl alcohol cleans magnetic bead, Elution Buffer elution, and obtained product is the full-length cDNA with specific molecular label.
Embodiment 3 uses the DNA banking process of transposase
(Vazyme) TruePrep DNA Library Prep produced using Nanjing Vazyme Biotechnology Co., Ltd.
Kit V2 for Illumina (TD503) kit carries out, and used reagent and material are provided by the kit, for example,
Swivel base enzyme buffer liquid, PCR buffer, high fidelity enzyme etc..
(Vazyme) Tn5 that transposase and embedding buffer are produced by Nanjing Vazyme Biotechnology Co., Ltd.
Transposome (S111) kit provides.
(1) connector embeds
Reaction system as shown in table 7 is configured, mixing is gently blown and beaten with pipettor, the reaction system prepared is placed in PCR
It is reacted 1.5 hours for 30 DEG C on instrument, obtains transposase embedding compound, -20 DEG C of preservations.
Table 7
(2) the full-length cDNA fragmentation with molecular label
1) each reactive component as shown in table 8 is successively added in sterilizing PCR pipe, and mixing is gently blown and beaten using pipettor.
Table 8
| Component | Volume |
| 1ng cDNA | 1μl |
| Swivel base enzyme buffer liquid | 4μl |
| Transposase embeds compound | 5μl |
| ddH2O | 40μl |
| Total | 50μl |
2) PCR reaction as shown in table 9 is carried out, 5 × TS of 5 μ l is added into reaction product immediately, gently using pipettor
Piping and druming mixes, and is placed at room temperature for 5min.
Table 9
| Heat lid | 105℃ |
| 55℃ | 10min |
| 10℃ | Hold |
(3) PCR is enriched with the 5 ' ends of cDNA
1) PCR reaction solution as shown in table 10 is configured, is mixed gently with pipettor, of short duration be collected by centrifugation is placed on ice.
Table 10
Wherein, the sequence of upstream primer N7 is as shown in SEQ ID NO.4: CAAGCAGAAGACGGCATACGAGATxxxxx
XxxGTCTCGTGGGCTCGG, wherein xxxxxxxx is index sequence；The sequence of downstream primer N5 is as shown in SEQ ID NO.5:
AATGATACGGCGACCACCGAGATCTACACxxxxxxxxTCGTCGGCAGCGTC, xxxxxxxx are index sequence.
2) program as shown in table 11 is run in PCR instrument:
Table 11
(3) PCR product purifies
The VAHTS DNA CleanBeads of (Vazyme) Biotechnology Co., Ltd production is only praised using Nanjing promise
(N411) PCR product is adsorbed, 80% ethyl alcohol cleans magnetic bead, Elution Buffer elution, and finally obtained product is richness
Collect the library at the 5 ' ends of cDNA.
Finally it should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although ginseng
It is described the invention in detail according to preferred embodiment, those skilled in the art should understand that, it can be to invention
Technical solution is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered
In scope of the presently claimed invention.
Sequence table
<110>Nanjing Vazyme Biotechnology Co., Ltd.
<120>the specific molecular label of cDNA a kind of and its application
<160> 6
<210>SEQ ID NO. 1
<211>25
<212>DNA
<213>artificial synthesized
AAGCAGTGGTATCAACGCAGAGTAC
<210>SEQ ID NO. 2
<211>34
<212>DNA
<213>artificial synthesized
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
<210>SEQ ID NO. 3
<211>61
<212>DNA
<213>artificial synthesized
AAGCAGTGGTATACGCAGAGTACGTTCGTGGGCTCGGTTCATAAGAGACAGNNNNNNNT30VN
<210>SEQ ID NO. 4
<211>39
<212>DNA
<213>artificial synthesized
CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG
<210>SEQ ID NO. 5
<211>43
<212>DNA
<213>artificial synthesized
AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC
<210>SEQ ID NO. 6
<211>29
<212>DNA
<213>artificial synthesized
CTCTTATACACATCTGACGCTGCCGACGA
Claims (6)
1. the specific molecular label of cDNA a kind of, which is characterized in that the nucleotide sequence such as SEQ of the specific molecular label
Shown in ID NO.3, particular sequence is as follows: AAGCAGTGGTATCAACGCAGAGTACGTCTCGTGGGCTCGGAGATGTGTATA
AGAGACAG(N)nT30VN, wherein N is random sequence, total n, n 5-10.
2. the specific molecular label of cDNA as described in claim 1 builds the application in library in cDNA.
3. the specific molecular label of cDNA as described in claim 1 builds the application in library in the cDNA interrupted using transposase.
4. a kind of cDNA banking process interrupted using transposase, which comprises the steps of:
1) separation is unicellular, will discharge total serum IgE after slender cellular lysate；Using mRNA as specific molecular described in template, claim 1
Label is that primer reverse transcription synthesizes the first chain cDNA, and the specific molecular label is added in the 5 ' ends of the first chain cDNA, and inverse
In transcription, using the template switch activity of reverse transcriptase in the 3 ' ends of the first chain cDNA plus the preceding paragraph joint sequence, this is connect
Header sequence with as universal primer sequence shown in SEQ ID NO.1 it is identical；Using the first chain cDNA as template, with such as SEQ IDNO.1
Shown in universal primer sequence be full-length cDNA of the primer by specific molecular label described in PCR amplification anamorphic zone, and carry out
Purifying；
2) full-length cDNA obtained by step 1) and transposase embedding compound are incubated for, complete cDNA fragmentation and adjunction head；
Wherein, the transposase embedding compound includes transposase and annealing connector；
3) PCR amplification is carried out to the resulting fragmentation products of step 2) using upstream primer N7 and downstream primer N5, is enriched with cDNA
5 ' end；Wherein, the sequence of upstream primer N7 is as shown in SEQ ID NO.4, the sequence of downstream primer N5 such as SEQ ID NO.5 institute
Show；
4) pcr amplification product obtained by step 3) is purified, obtains 5 ' the end texts of the cDNA with the specific molecular label
Library.
5. the cDNA banking process according to claim 4 interrupted using transposase, which is characterized in that in step 2), institute
The nucleotide sequence of the annealing connector in transposase embedding compound is stated as shown in SEQ ID NO.6；The transposase is Tn5
Family's transposase.
6. the cDNA banking process according to claim 4 or 5 interrupted using transposase, which is characterized in that in step 2),
The cDNA fragmentation and plus jointing method include: preparation reaction mixture: the transposase embedding of claim 4 or 5 is multiple
Close object 3-7 μ l, swivel base enzyme reaction buffer solution 4-8 μ l；Full-length cDNA is mixed with reaction mixture, is blown and beaten and is mixed with pipettor；
The 50-58 DEG C of reaction 10-15min in PCR instrument.
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