CN108060460A - A kind of library construction and sequencing approach of small RNA - Google Patents
A kind of library construction and sequencing approach of small RNA Download PDFInfo
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Abstract
This application discloses a kind of library constructions and sequencing approach of small RNA.The banking process of the application includes：(1) small RNA segments are connected with 3 ' connector of CG libraries；(2) step (1) product is connected with 5 ' connector of CG libraries；(3) by step (2) product reverse transcription into cDNA；(4) PCR amplification, purifying recycling pcr amplification product are carried out to cDNA；(5) single-stranded separation is carried out to purifying recycling pcr amplification product, single stranded DNA is cyclized；(6) cyclisation product is digested, recycled, obtain small RNA CG libraries.The banking process of the application, new CG libraries, which are provided, for small RNA builds storehouse scheme, so that the horizontal small RNA sequencings of full-length genome based on CG sequencings are achieved, excavated for new small RNA molecules, cancer microRNA target prediction and identification, sample room Differential expression analysis, small RNA clusters and expression pattern analysis etc. are laid a good foundation, also provide scientific basis for early diagnosis of cancer and targeted therapy detection.
Description
Technical field
This application involves small RNA to build storehouse and sequencing field, more particularly to a kind of library construction side of small RNA
Method and sequencing approach.
Background technology
In recent years epidemiologic data is shown, 600,000 people of cases of lung cancer is newly sent out in China, for malignant tumour new cases then
19.59%, occupy malignant tumour first place.Even if in the modern times of pharmaceutical science prosperity, 5 years survival rates of lung cancer patient are especially removed
5 years survival rates of the patients with solid tumor beyond patients with blood cancer are less than 50%.In all cancer patients, about 2/3 is late examined
Disconnected, most of is dead in two years after cancer diagnosis.The result of this clumsiness in treatment of cancer is not only therapy
Problem, it is often more important that, on the one hand, the early diagnosis of cancer is extremely difficult, and even cancer of late stage, which will be realized, accurately makes a definite diagnosis
Also it is not easy；On the other hand, the prognosis after remedy measures are taken and tracking (the i.e. so-called follow- to cancer patient
Up), also it is not easy very much.
Small RNA are a major class regulatory molecules, are almost present in all organisms.Small RNA include：
MiRNA, ncRNA, siRNA, snoRNA, piRNA, rasiRNA etc..Small RNA pass through diversified action pathway, bag
MRNA degradations, Translational repression, heterochromatin formation and DNA removals etc. are included, to regulate and control the growth and development of organism and disease hair
It is raw.
There is very big difference, and specific small RNA in expression of the Small RNA between cancerous tissue and non-cancer tissue
Abnormal expression is related to specific cancer, thus small RNA can be used as the molecular labeling of diagnosis cancer, such as derive from
The miR-21 of ectosome (exosome) can be as the prognostic indicator of several cancers, and miR-21 is related to miR-31 and cancer of the esophagus.This
Outside, if can control miRNA in ectosome expression and release or using ectosome carry send some tools it is medicative
MiRNA equimoleculars can then carry out certain cancers and the targeted therapy of disease to target cell.By building small RNA libraries
And large-scale sequencing analysis are carried out to small RNA libraries, it can therefrom obtain the small of species full-length genome level
RNA collection of illustrative plates realizes the excavation for including new small RNA molecules, the prediction and identification of the target gene that works, sample room differential expression
Analysis, the scientific applications such as Small RNA clusters and expression pattern analysis.
With the development of two generation sequencing technologies, Roche GS FLX Titanium, Illumina Solexa GA IIx and
AB SOLID 4 can carry out large scale sequencing analysis, Illumina Solexa GA IIx and ABI to small RNA
The reading length of Solid has just coordinated the short sequence of small RNA and flux is big, can obtain higher coverage rate, Illumina
Solexa GA IIx extensive uses in small RNA sequencings.
Complete genome group (Complete Genomics, abridge CG) sequencing is a kind of two new generation sequencing technologies, it is used
High-density DNA nano chips technology, the intercalation of DNA nanosphere on chip, use is discontinuous, non-chain joint probe anchoring (cPAL)
Technology reads sequence, has higher flux, and the expense of each gene order-checking is down to 1000 dollars.In short, based on CG texts
The CG sequencing technologies of storehouse structure have the characteristics that build that storehouse is at low cost, easy to operate, the time is short, it is high to build Kucheng's power, it is fast to be sequenced, and have
Very high promotional value.At present, there has been no what is be sequenced on small RNA CG library constructions and small RNA CG related to grind
Study carefully and report；Therefore, the banking process that a kind of CG suitable for small RNA is sequenced is studied, for large-scale small RNA
Sequencing and the further investigation and exploitation of small RNA are of great significance.
The content of the invention
The purpose of the application is to provide a kind of library constructing method of new small RNA, for small RNA CG texts
The kit in storehouse and the sequencing approach based on small RNA library constructions are built in storehouse.
The application employs following technical scheme：
The one side of the application discloses a kind of library constructing method of small RNA, comprises the following steps；
(1) small RNA segments are connected with the 3 ' connectors in CG libraries；
(2) product of step (1) is connected with the 5 ' connectors in CG libraries；
(3) by the product reverse transcription of step (2) into cDNA；
(4) PCR amplification is carried out to the cDNA that step (3) obtains, and purifies recycling pcr amplification product；
(5) single-stranded separation is carried out to purifying recycling pcr amplification product, and the single stranded DNA of acquisition is cyclized；
(6) product of single stranded DNA cyclisation is digested, and recycles the product after digestions, that is, obtain small
RNA CG libraries.
It should be noted that the library constructing method of the small RNA of the application, compared to general DNA CG libraries
Banking process has following improvement and difference：1) structure in DNA CG libraries needs to carry out dephosphorylation and end to building storehouse segment
Filling-in processing is held, and the structure in the small RNA CG libraries of the application is without this step；2) constructing plan in DNA CG libraries
In 3 ' and 5 ' connectors be in same reaction system, while be connected to the both ends for building storehouse segment, and the small RNA of the application
3 ' and 5 ' connector of structure in library is that two reaction systems is divided to be connected respectively to the both ends for building storehouse segment, also, the one of the application
3 ' and 5 ' connectors employed in kind realization method are that the applicant synthesizes particular for small RNA autonomous Designs；3)DNA
The structure in CG libraries, which connects, needs after connector the reaction for carrying out nick translation at once to carry out PCR amplification again, and the small of the application
The structure in RNA CG libraries need not carry out the reaction of nick translation, need to will only connect the library fragments of connector after reverse transcription again into
Row PCR amplification；4) structure in DNA CG libraries can carry out single-stranded separating treatment after PCR amplification, and the application
The structure in small RNA CG libraries carries out single-stranded point again after then need to recycling pcr amplification product first with Native PAGE glue purification
From processing.
Wherein, 3 ' and 5 ' connectors are divided to two reaction systems to be connected respectively to the both ends for building storehouse segment, are because of the application's
The reason for sequence of 3 ' end connectors also acts as sequence label first allows target sequence and 3 ' that reaction is held to may insure the two reaction more
Add fully, avoid causes subsequently to expand since sequence label caused by the influence of 5 ' end connectors cannot be combined fully with target sequence
A large amount of unqualified sequences are generated during increasing.The application recycles pcr amplification product using Native PAGE glue purification, is because in PCR
There is very big gap in the pcr amplification product allele clip size obtained after amplification, if do not purified, may exist very much
The amplified production of sequence label is not contained, these cannot be used for subsequent experiment.And the resolution of general agarose gel electrophoresis
Rate is low, can only distinguish the DNA fragmentation of 0.5-25kb, the molecular weight very little of small RNA, agarose gel electrophoresis is likely to can not
Separation, therefore the application has especially selected the higher Native PAGE glue purification of resolution ratio.It is but big to molecular weight when building library
It is small not to be strict with, it need to only extract DNA molecular, therefore common purifying.
Preferably, in step (4), purifying recycling pcr amplification product is using Native PAGE glue；In step (6), return
The product after digestions is received, using paramagnetic particle method.
The another side of the application discloses a kind of kit that storehouse is built for small RNA CG libraries, including CG libraries 3 '
5 ' connector of connector and CG libraries；3 ' connector of CG libraries is sequence shown in SEQ ID NO.1, and 5 ' connector of CG libraries is SEQ ID
Sequence shown in NO.2；
SEQ ID NO.1:
5’-GTCTCCAGTCGAAGCCCGATCnnnnnnnnnnGAGCTTGTC-3’
SEQ ID NO.2:5’-UCCUAAGACCGCUUGGCCUCCGACUU-3’
Wherein, what n was repeated is selected from A, G, C or T.
It should be noted that in the 3 ' connector of CG libraries of sequence shown in SEQ ID NO.1, " nnnnnnnnnn " represents 10bp
Barcode sequences.3 ' the connector of CG libraries and 5 ' connector of CG libraries of the application is designed particular for small RNA, can
To be suitable for small RNA CG library constructions well.
Preferably, 3 ' connector of CG libraries is at least one of for following four nucleotide fragments, four kinds of nucleotide fragments according to
Sequence is sequence shown in SEQ ID NO.6 to SEQ ID NO.9.
SEQ ID NO.6：
5’-GTCTCCAGTCGAAGCCCGATCATAAGGCAGTGAGCTTGTCT-3’
SEQ ID NO.7：
5’-GTCTCCAGTCGAAGCCCGATCTTGATAGATTGAGCTTGTCT-3’
SEQ ID NO.8：
5’-GTCTCCAGTCGAAGCCCGATCCCTTCCTGGTGAGCTTGTCT-3’
SEQ ID NO.9：
5’-GTCTCCAGTCGAAGCCCGATCAATATCTCTCGAGCTTGTCT-3’
It should be noted that four 3 ' connectors of CG libraries of sequence shown in SEQ ID NO.6 to SEQ ID NO.9, simply
In a kind of realization method of the application, specifically used for the small RNA in the ectosome in Serum of Patients with Lung Cancer source four
The connector of Barcode sequences, however not excluded that others Barcode sequences can also be used.
Preferably, the kit of the application further includes reverse transcription primer, and reverse transcription primer is sequence shown in SEQ ID NO.3
Row,
SEQ ID NO.3:
5’-AGACAAGCTCnnnnnnnnnnGATCGGGCTTCGACTGGAGAC-3’
Wherein, what n was repeated is selected from A, G, C or T.
It should be noted that in the reverse transcription primer of sequence shown in SEQ ID NO.3, " nnnnnnnnnn " represents 10bp's
Barcode sequences.
Preferably, reverse transcription primer is at least one of following four primer, and four kinds of primers are sequentially SEQ ID
Sequence shown in NO.10 to SEQ ID NO.13.
SEQ ID NO.10：
5’-AGACAAGCTCACTGCCTTATGATCGGGCTTCGACTGGAGAC-3’
SEQ ID NO.11：
5’-AGACAAGCTCAATCTATCAAGATCGGGCTTCGACTGGAGAC-3’
SEQ ID NO.12：
5’-AGACAAGCTCACCAGGAAGGGATCGGGCTTCGACTGGAGAC-3’
SEQ ID NO.13：
5’-AGACAAGCTCGAGAGATATTGATCGGGCTTCGACTGGAGAC-3’。
It should be noted that four reverse transcription primers of sequence shown in SEQ ID NO.10 to SEQ ID NO.13, simply
In a kind of realization method of the application, specifically used for the small RNA in the ectosome in Serum of Patients with Lung Cancer source four
The reverse transcription primer of Barcode sequences, however not excluded that others Barcode sequences can also be used.
Preferably, the kit of the application further includes cDNA amplimers, and cDNA amplimers is shown in SEQ ID NO.4
Sequence,
SEQ ID NO.4:5’-TCCTAAGACCGCTTGGCCTCCGACTT-3’。
Preferably, the kit of the application further includes is cyclized nucleotide fragments, subring for the auxiliary of single stranded DNA cyclisation
Change nucleotide fragments are sequence shown in SEQ ID NO.5,
SEQ ID NO.5:5’-TCGAGCTTGTCTTCCTAAGACCGC-3’。
It should be noted that auxiliary cyclisation nucleotide fragments are actually the Splint that single stranded DNA is aided in be cyclized
Segment, principle are that the both ends of single stranded DNA are hybridized to respectively in Splint segments, using Splint segments as template, in ligase
Under the action of, the both ends of single stranded DNA are connected into cyclization, realize single stranded DNA cyclisation.
Preferably, various reagents used by can also including building storehouse in the kit of the application in the process, for example, connector
Coupled reaction reagent, Reverse Transcription, cDNA amplifing reagents, single stranded DNA cyclization reagent etc., certainly, these reagents can also lead to
Commercially available purchase is crossed, is not specifically limited herein.
The banking process for disclosing the application on one side again of the application or the kit of the application, horizontal in full-length genome
Application in the preparation of small RNA collection of illustrative plates or analysis.
It should be noted that the banking process and kit of the application, inherently for complete genome group small RNA
, therefore, the small RNA collection of illustrative plates that can be used for full-length genome level is prepared or analyzed.And horizontal based on full-length genome
Small RNA atlas analysis can carry out the excavation of new small RNA molecules, the prediction of cancer target gene or identification, sample room
Differential expression analysis, small RNA clusters and expression pattern analysis etc., are not specifically limited herein.
The banking process for disclosing the application on one side again of the application or the kit of the application are examined in early days preparing cancer
Application in the reagent of disconnected or targeting cancer therapy detection.
It is appreciated that the banking process and kit of the application can prepare the small RNA collection of illustrative plates of full-length genome level,
And the prediction or identification of cancer target gene can be carried out based on this, therefore, the banking process and kit of the application completely can be with
It is detected for early diagnosis of cancer or targeting cancer therapy, and then can be used for preparing early diagnosis of cancer or targeting cancer therapy
The reagent of detection.
The application's discloses a kind of sequencing approach of small RNA on one side again, the library constructing method including the application,
Library construction is carried out, then constructed library is sequenced using CG microarray datasets.
The advantageous effect of the application is：
The library constructing method of the application small RNA provides a kind of new CG libraries particular for small RNA and builds
Storehouse scheme so that the small RNA sequencings of the full-length genome level based on CG sequencings are achieved, and then for based on full-length genome
The new small RNA molecules of horizontal small RNA atlas analysis are excavated, cancer microRNA target prediction and identification, sample room difference table
It lays a good foundation up to scientific applications such as analysis, small RNA clusters and expression pattern analysis, while is also early diagnosis of cancer and cancer
The detection of disease targeted therapy provides favourable scientific basis.
Description of the drawings
Fig. 1 is the FB(flow block) of the banking process in small RNA CG libraries in the embodiment of the present application.
Specific embodiment
Complete genome group (Complete Genomics, abridge CG) sequencing is a kind of two new generation sequencing technologies, at present
Storehouse scheme is built through there are a DNA CG libraries of comparative maturity, still, there has been no on small RNA CG library constructions and small
The correlative study of RNA CG sequencings and report.
In view of CG sequencing technologies, which have, builds the spies such as storehouse is at low cost, easy to operate, the time is short, it is high to build Kucheng's power, sequencing is fast
Point, what the application especially researched and developed and proposed a kind of new CG libraries for small RNA builds storehouse scheme, also, in this Shen
In embodiment please, especially have developed for the 3 ' connectors of small RNA and 5 ' connectors and subsequently corresponding a series of anti-
Transcription primers, for the cDNA of reverse transcription carry out PCR amplification cDNA amplimers and for single stranded DNA cyclisation it is auxiliary
Help cylic nucleotide segment so that the structure in small RNA CG libraries is achieved.According to above research, the application is also into one
Step proposes a kind of kit that storehouse is built for small RNA CG libraries, can simply and easily carry out small RNA CG texts
The structure in storehouse.
The application is described in further detail below by specific embodiment.Following embodiment is only to the application into traveling
One step illustrates, should not be construed as the limitation to the application.
Embodiment
Storehouse and sequencing approach are built this example provides a kind of new small RNA, the small RNA available for all sources
The structure in library, the small RNA CG libraries of structure are suitable for second generation sequencing technologies complete genome group (Complete
Genomics, i.e. CG) microarray dataset.
The banking process in the small RNA CG libraries of this example, as shown in Figure 1, comprising the following steps；
(1) small RNA segments are connected with the 3 ' connectors in CG libraries；
(2) product of step (1) is connected with the 5 ' connectors in CG libraries；
(3) by the product reverse transcription of step (2) into cDNA；
(4) PCR amplification is carried out to the cDNA that step (3) obtains, and purifies recycling pcr amplification product；
(5) single-stranded separation is carried out to purifying recycling pcr amplification product, and the single stranded DNA of acquisition is cyclized；
(6) product of single stranded DNA cyclisation is digested, and recycles the product after digestions, that is, obtain small
RNA CG libraries.
Wherein, in step (4), using Native PAGE glue, this example specifically uses purifying recycling pcr amplification product
6%PAGE glue is recycled；In step (6), the product after recycling digestions, using paramagnetic particle method, this example specifically uses
PEG32beads。
In the banking process in the small RNA CG libraries of this example, it has been specifically designed and has been connect for the 3 ' of small RNA segments
Head connection is connected with 5 ' connectors and corresponding reverse transcription primer, cDNA amplimers and the subring for single stranded DNA cyclisation
Change nucleotide fragments.
3 ' connector of CG libraries is sequence shown in SEQ ID NO.1, and 5 ' connector of CG libraries is sequence shown in SEQ ID NO.2,
Reverse transcription primer is sequence shown in SEQ ID NO.3, and cDNA amplimers are sequence shown in SEQ ID NO.4, and auxiliary is cyclized core
Acid fragments are sequence shown in SEQ ID NO.5；
SEQ ID NO.1:
5’-GTCTCCAGTCGAAGCCCGATCnnnnnnnnnnGAGCTTGTC-3’
SEQ ID NO.2:5’-UCCUAAGACCGCUUGGCCUCCGACUU-3’
SEQ ID NO.3:
5’-AGACAAGCTCnnnnnnnnnnGATCGGGCTTCGACTGGAGAC-3’
SEQ ID NO.4:5’-TCCTAAGACCGCTTGGCCTCCGACTT-3’
SEQ ID NO.5:5’-TCGAGCTTGTCTTCCTAAGACCGC-3’
In more than sequence, what n was repeated is selected from A, G, C or T, and " nnnnnnnnnn " represents the Barcode sequences of 10bp.
According to the banking process in more than small RNA CG libraries, this example is specifically to the outer of 4 Serum of Patients with Lung Cancer sources
Small RNA in body have carried out CG libraries Jian Ku.First with the ExoQuickTM Exosomes of SBI companies
Precipitation Solution are enriched with the ectosome in serum, and the SearMir kit of SBI companies is then recycled to take out
Carry the small RNA in ectosome, the concentration of four small RNA that this example specifically extracts is 0.162-0.424ng/ μ L, volume
It is 30 μ L.
3 ' connector of CG libraries is sequence shown in SEQ ID NO.1, wherein, " nnnnnnnnnn " represents the Barcode of 10bp
For distinguishing the sequence of different samples during the upper machines sequencing of sequence, i.e. multiple samples mixing, this example is for 4 patients with lung cancer
Small RNA have used the 3 ' connectors of 4 band 10bp Barcode, and the 3 ' connectors of 4 band 10bp Barcode are sequentially SEQ
Sequence shown in ID NO.6 to SEQ ID NO.9,
SEQ ID NO.6：
5’-GTCTCCAGTCGAAGCCCGATCATAAGGCAGTGAGCTTGTCT-3’
SEQ ID NO.7：
5’-GTCTCCAGTCGAAGCCCGATCTTGATAGATTGAGCTTGTCT-3’
SEQ ID NO.8：
5’-GTCTCCAGTCGAAGCCCGATCCCTTCCTGGTGAGCTTGTCT-3’
SEQ ID NO.9：
5’-GTCTCCAGTCGAAGCCCGATCAATATCTCTCGAGCTTGTCT-3’。
Reverse transcription primer is sequence shown in SEQ ID NO.3, wherein, " nnnnnnnnnn " represents the Barcode sequences of 10bp
Row, this example have used the reverse transcription primer of 4 band 10bp Barcode, 4 bands for the small RNA of 4 patients with lung cancer
The reverse transcription primer of 10bp Barcode is sequentially sequence shown in SEQ ID NO.10 to SEQ ID NO.13,
SEQ ID NO.10：
5’-AGACAAGCTCACTGCCTTATGATCGGGCTTCGACTGGAGAC-3’
SEQ ID NO.11：
5’-AGACAAGCTCAATCTATCAAGATCGGGCTTCGACTGGAGAC-3’
SEQ ID NO.12：
5’-AGACAAGCTCACCAGGAAGGGATCGGGCTTCGACTGGAGAC-3’
SEQ ID NO.13：
5’-AGACAAGCTCGAGAGATATTGATCGGGCTTCGACTGGAGAC-3’。
The small RNA of 4 patients with lung cancer use identical 5 ' connector of CG libraries, i.e., sequence shown in SEQ ID NO.2
5 ' connector of CG libraries；Using identical cDNA amplimers, i.e., the cDNA amplimers of sequence shown in SEQ ID NO.4；Using
Identical auxiliary cyclisation nucleotide fragments, i.e., the auxiliary cyclisation nucleotide fragments of sequence shown in SEQ ID NO.5.
Small RNA in the ectosome in 4 Serum of Patients with Lung Cancer sources of this example have carried out the specific reaction that storehouse is built in CG libraries
System and condition are as follows：
3 ' the connectors in 1.small RNA segments connection CG libraries
The small RNA of 5 μ L nuclease frees of extraction with 10 μM of 3 ' connector of CG libraries, 1 μ L are mixed first, are made mixed
Close sample.Biased sample is positioned on ice immediately in Thermal cycler after 70 DEG C of reaction 2min, it is spare.
Then the reaction solution of 5 μ L of total volume is prepared, including：10 × T4 RNA connections buffer solution, 1 μ L, 1 × 50%PEG8000
(BioLabs) 2.5 μ L, 40U/ μ L RNase inhibitor (invitrogen) 0.5 μ L, 200U/ μ L T4 RNA ligase
1 μ L of 2truncated (BioLabs) amount to 5 μ L.
The reaction solution of 5 μ L is added to mixing in the biased sample being placed on ice, reacts 2 for 25 DEG C in Thermal cycler
Hour, then 12 DEG C of holdings.Complete the connection of 3 ' connectors.
After the connection of 3 ' connectors, reverse transcription primer annealing is connected to 3 ' in advance and connect by subsequent reverse transcription for convenience, this example
On head, specifically, 100 μM of 0.5 μ L of reverse transcription primer are added in 3 ' connector connection products, mixing, then Thermal
75 DEG C of reactions 5min, 37 DEG C of reactions 30min, 25 DEG C of reaction 15min in cycler.3 ' connector connection products after being annealed.
5 ' the connectors in 2.small RNA segments connection CG libraries
It takes the PCR pipe of new RNase-free, adds in the 5 ' connector of CG libraries of 10 μM of 1 μ L, 70 in Thermal cycler
DEG C reaction 2min, be positioned over immediately after on ice, it is spare.
Then the reaction solution of 2.5 μ L of total volume is prepared, including：1 μ L of 10mM ATP, 10U/ μ L T4 RNA ligases 1
(BioLabs) 1 μ L, 40U/ μ L RNase inhibitor (invitrogen) 0.5 μ L amount to 2.5 μ L.
The reaction solution of 2.5 μ L is added to mixing in the 5 ' connector of CG libraries being placed on ice, is then added mixture to
In 3 ' connector connection products after annealing, when 20 DEG C of reactions 1 are small in Thermal cycler.Complete the connection of 5 ' connectors.
3. reverse transcription generates cDNA
The reaction solution of 10.5 μ L of total volume is prepared, including：5×first stand buffer(invitrogen)5μL、
0.5 μ L of 0.1M DTT, 0.5 μ L of 10mM dNTP, 40U/ μ L RNase inhibitor (invitrogen)
0.5μL、200U/μLII Reverse Transcriptase (invitrogen) 0.5 μ L, nothing
3.5 μ L of nuclease water amount to 10.5 μ L.
The 10.5 μ L reaction solutions prepared are added in 5 ' connector connection products, mixing, 42 DEG C in Thermal cycler
React 40min, 70 DEG C of reaction 15min, then 12 DEG C of holdings.Obtain reverse transcription product cDNA.
The PCR amplification of 4.cDNA
First, the PCR reaction solution of 25 μ L of total volume is prepared, including：10×PfxAmplification Buffer
(invitrogen)5μL、50mM MgSO4 2μL、10mM dNTPs 2μL、2.5U/μL Platinum Pfx DNA
0.8 μ L of polymerase (invitrogen), 14.2 μ L of nuclease-free water, 20 μM of 1 μ L of cDNA amplimers amount to 25 μ L.
The PCR reaction solution of prepared 25 μ L is added in reverse transcription product, 50.5 μ L systems are amounted to, in PCR instrument
It is reacted as follows：
94 DEG C of 2min, subsequently into 20 Xun Huans：94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 50s, after circulation terminates, 72 DEG C
10min, 12 DEG C of holdings.
PCR reaction products are recycled using 6% Native PAGE glue purification, specific as follows：
(1) 10 μ L 6 × loading buffer mixings, 180V constant pressure electrophoresis are added in PCR product, this example uses 20bp
2 μ L of DNA ladder loadings are as marker；
(2) casing is made：Aperture is pricked with EP bottom of the tube of the syringe needle in 500 μ L is burnt, is then placed in 2mL EP pipes
In, and twined the lid of 2mL pipes and lid and tube body connecting place with preservative film；
(3) the target fragment PAGE glue cut is put into and pricked in the EP pipes of foraminate 500 μ L, room temperature 12000rpm centrifugations
2min crushes PAGE glue block；
(4) 0.3M NaCl are added in broken PAGE glue block, then 350rpm in constant temperature blending instrument, 25 DEG C, 2 it is small when；
(5) mixed liquor after mixing is transferred in 0.45 μm of filter column of spin-x, 4 DEG C, 13000rpm centrifugations 5min；
(6) the 3M sodium acetates of 1/10 volume, the glycogen of 2 μ L 5mg/ml, the nothing of 2-3 times of volume are sequentially added in filtrate
Water-ethanol, when then -80 DEG C of precipitations 1 are small；
(7) 4 DEG C, 13000rpm centrifuges 30min, and then addition 80% ethyl alcohol of 1mL gently bounces precipitation and washed；
(8) 4 DEG C, 13000rpm centrifugation 5min, room temperature adds in 22 μ L elution buffer dissolving precipitations after drying；I.e.
Obtain PCR product after purification.
PCR product after being recycled using Qubit dsDNA HS assay kit to purifying carries out concentration mensuration, as a result shows
Show, the purifying of 4 patients with lung cancer of this example, which is recycled after PCR product concentration can be used for, continues storehouse.
5. single-stranded separation
(1) by the PCR product after purification of acquisition add 1 × TE to total volume be 60 μ L；
(2) following reagent is prepared in advance：100%Tween 20 is diluted to 0.5%Tween 20, Streptavidin
Beads vortex mixings；
(3) shift to an earlier date 15min and configure 1 × BBB/0.5%Tween20Mix, 1 × BWB/0.5%Tween20Mix, 0.1M
NaOH, collocation method are as follows：
1 × BBB/0.5%Tween20Mix of 30.3 μ L includes：1 × BBB, 30 μ L, 0.5%Tween200.3 μ L；Its
In, 1 × BBB includes：Bead Binding Buffer, 110mM Tris-HCl and 200mM NaCl；
1 × BWB/0.5%Tween20Mix of 2020 μ L includes：1 × BWB, 2000 μ L, 0.5%Tween20 20 μ L；Its
In, 1 × BWB includes：Bead Wash Buffer, 10mM Tris-HCl and 40mM NaCl.
The 0.1M NaOH of 26 μ L add 20.8 μ L of water to be formulated by the 5.2 μ L of NaOH of 0.5M.
(4) the Streptavidin Beads being connected with biotin B are washed with the following method
A. each sample takes 30 μ L Streptavidin Beads to be added in 1.5mL EP pipes, then adds 4 times of bodies
1 long-pending × BBB, mixing are placed on staticaccelerator adsorption on magnetic frame, adjust the direction of EP pipes so that beads is in 1 × BBB washing lotions
Front and rear travelling, that is, rotate EP pipes 2 times, after abandoning supernatant, repeats aforesaid operations once；
B. take out not collophore and add in 1 times of volume, i.e. 30 μ L, 1 × BBB/0.5%Tween20Mix suspensions, room temperature after mixing
It stands；
(5) 4 × BBB mixings of 20 μ L are added in into the PCR product sample of 60 μ L, step is then transferred into and contains 30 μ L
1 × BBB/0.5%Tween20Mix dissolving beads not collophore in mixing, this 110 μ L mixture at room temperature combine 15-
20min, centre gently play it is even once
(6) above-mentioned not collophore magnetic frame is placed into 3-5min, abandoning supernatant, with 1 × BWB/0.5% of 1mL
Tween20Mix is washed 2 times, and method is the same as the washing methods of Streptavidin Beads；
(7) 26 μ L 0.1M NaOH are added in into above-mentioned beads, 10min is placed after blowing and beating mixing, then is placed on magnetic frame
3-5min takes supernatant into new PCR pipe；
(8) 13 μ L 0.3M MOPS are added in into above-mentioned PCR pipe, mixing is spare, this step products can be frozen in -20
℃；
Obtain separated Single-stranded DNA fragments.
6. the cyclisation of single stranded DNA
20 μM of the auxiliary cyclisation nucleotide fragments of 10 μ L are added in the single stranded DNA sample of the 39 μ L obtained one step up,
Biased sample is made.
Then the reaction solution of 11 μ L of total volume is prepared, including：10×TA Buffer(LK1)6μL、100mM ATP 0.6μ
L, 0.2 μ L of 600U/ μ L ligases, 4.2 μ L of water amount to 11 μ L.
It centrifuges, is then added in the biased sample of 49 μ L after the reaction solution of 11 μ L is shaken abundant mixing, concussion 10s is mixed
It is even, after brief centrifugation, when 37 DEG C of reactions 1.5 are small.Obtain cyclisation product.
7. the not cyclized single stranded DNA of digestions
The reaction solution of 5 μ L of total volume is prepared, including：10x TA Buffer(LK1)1μL、20U/μL ExoI 3μL、
1 μ L of 200U/ μ L ExoIII amount to 5 μ L.
It centrifuges, is then added in the cyclisation product of previous step after the reaction solution of 5 μ L is shaken abundant mixing, shake 10s
Mixing, 37 DEG C of reaction 30min after brief centrifugation.
Digestion 30min completes to add in 2.5 μ L 500mM EDTA termination endonuclease reactions in backward reaction product, obtains digestion
Reaction product.
8. utilize the product after PEG32beads purifying recycling digestions
(1) above-mentioned endonuclease reaction product is transferred to new 1.5mL not in collophore, adds in the PEG32beads/ of 84.5 μ L
During which 0.5%Tween20, room temperature combination 15min blow and beat mixing once, in wherein PEG32beads/0.5%Tween20,
PEG32beads:0.5%Tween20=100:1；
(2) collophore is not placed in supernatant discarding after magnetic frame 3-5min；
75% ethyl alcohol of (3) 700 μ L washes twice, and inverts collophore front-rear direction during washing so that beads is in ethyl alcohol
Middle travelling, every time washing travelling 2-3 times；
(4) dry at room temperature and add in 27 μ L TE/0.5%Tween20, room temperature combination 15min, intermediate mixing is once；Its
In, in TE/0.5%Tween20, TE:0.5%Tween20=500:1；
(5) supernatant is transferred in new 1.5mL EP pipes, finally obtains the small RNA CG texts that product is this example
Storehouse.
Using QubitTMSsDNA Assay Kit are quantitative to carry out library Quality Control and concentration mensuration, Buffer and dyestuff ratio
Example is 199:1, votex and mixing for standby use is centrifuged after mixing, and dyestuff working solution is separately added into 10 μ L's after taking two part of 190 μ L dilution
Two kinds of standard items votex simultaneously centrifuge mixing for standby use, take 198 μ L dilute after dyestuff working solution add in 2 μ L samples, after votex and from
The heart carries out Qubit instrument quantitatives.The results show that the small RNA CG libraries of this example structure can be used for the survey of CG microarray datasets
Sequence.
The small RNA CG libraries of this example are sequenced, and lower machine data are analyzed, it is specific as follows：
Machine is sequenced in the CG microarray datasets of 1.small RNA CG libraries
In library construction process, the 3 ' connectors added by small RNA point in the ectosome in 4 Serum of Patients with Lung Cancer sources
Barcode that Bao Han be not different, therefore can be by the small RNA's of this four separate sources in PAGE glue recycling step
Pcr amplification product, which is put together, to be recycled, and eventually forming one has 4 differences initial sample pooling together
CG mixing library.QubitTMSsDNA Assay Kit quantify testing result and show, the concentration in mixing CG 1adapter libraries
More than 7.5fmol/ μ L, volume is 20 μ L, reaches the concentration requirement that machine is sequenced on CG libraries, general CG libraries 1adapter sequencings
Make DNB need 120fmol.The mixing library is sequenced using CG 1adapter 12+19 both-ends, surveys 2 lane, 7.5G/
lane。
The analysis of machine data under 2.small RNA CG libraries.
CG mixing library carries out data analysis after sequencing, and analysis result is shown, mixes 4 Serum of Patients with Lung Cancer in library
The data volume that small RNA CG libraries in the ectosome in source are sequenced is all higher than 60M reads, and data are analyzed through comparing
It was found that 55 known small RNA, and wherein include 36 and the relevant small RNA of cancer.It should be the result shows that source
Small RNA in serum ectosome may be employed that at low cost, easy to operate, the time is short, builds the high CG of Kucheng's power
1adapter builds storehouse mode and is built into CG libraries, builds successful CG libraries suitable for the fast complete base of sequencing throughput height, sequencing
Because of a group CG microarray datasets.The machine data analysis under to the Small RNA CG libraries by way of building storehouse sequencing, can obtain with
The information of the relevant small RNA of cancer can be that the early diagnosis of cancer and the follow-up personalized targeted therapy of development are established
Fixed basis.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, it is impossible to assert this Shen
Specific implementation please is confined to these explanations.For those of ordinary skill in the art to which this application belongs, do not taking off
On the premise of conceiving from the application, several simple deduction or replace can also be made.
SEQUENCE LISTING
<110>Shenzhen Hua Da life science institute
<120>A kind of library construction and sequencing approach of small RNA
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Claims (10)
1. a kind of library constructing method of small RNA, it is characterised in that：Comprise the following steps；
(1) small RNA segments are connected with the 3 ' connectors in CG libraries；
(2) product of step (1) is connected with the 5 ' connectors in CG libraries；
(3) by the product reverse transcription of step (2) into cDNA；
(4) PCR amplification is carried out to the cDNA that step (3) obtains, and purifies recycling pcr amplification product；
(5) single-stranded separation is carried out to purifying recycling pcr amplification product, and the single stranded DNA of acquisition is cyclized；
(6) product of single stranded DNA cyclisation is digested, and recycles the product after digestions, that is, obtain the small
RNA CG libraries.
2. library constructing method according to claim 1, it is characterised in that：In the step (4), purifying recycling PCR expands
Increase production object using Native PAGE glue；In the step (6), the product after recycling digestions, using paramagnetic particle method.
3. a kind of kit that storehouse is built for small RNA CG libraries, it is characterised in that：Including 3 ' connector of CG libraries and CG texts
5 ' connector of storehouse；
3 ' the connector of CG libraries is sequence shown in SEQ ID NO.1, and the 5 ' connector of CG libraries is sequence shown in SEQ ID NO.2
Row；
SEQ ID NO.1:
5’-GTCTCCAGTCGAAGCCCGATCnnnnnnnnnnGAGCTTGTC-3’
SEQ ID NO.2:5’-UCCUAAGACCGCUUGGCCUCCGACUU-3’
Wherein, what n was repeated is selected from A, G, C or T.
4. kit according to claim 3, it is characterised in that：3 ' the connector of CG libraries is following four nucleotide piece
At least one of section, four kinds of nucleotide fragments are sequentially sequence shown in SEQ ID NO.6 to SEQ ID NO.9.
5. the kit according to claim 3 or 4, it is characterised in that：The kit further includes reverse transcription primer, described
Reverse transcription primer is sequence shown in SEQ ID NO.3,
SEQ ID NO.3:
5’-AGACAAGCTCnnnnnnnnnnGATCGGGCTTCGACTGGAGAC-3’
Wherein, what n was repeated is selected from A, G, C or T.
6. kit according to claim 5, it is characterised in that：The reverse transcription primer be following four primer in extremely
Few one kind, four kinds of primers are sequentially sequence shown in SEQ ID NO.10 to SEQ ID NO.13.
7. the kit according to claim 3 or 4, it is characterised in that：The kit further includes cDNA amplimers, institute
CDNA amplimers are stated as sequence shown in SEQ ID NO.4,
SEQ ID NO.4:5’-TCCTAAGACCGCTTGGCCTCCGACTT-3’。
8. the kit according to claim 3 or 4, it is characterised in that：The kit further includes to be cyclized for single stranded DNA
Auxiliary cyclisation nucleotide fragments, it is described auxiliary cyclisation nucleotide fragments be sequence shown in SEQ ID NO.5,
SEQ ID NO.5:5’-TCGAGCTTGTCTTCCTAAGACCGC-3’。
9. library constructing method according to claim 1 or 2 or claim 3-8 any one of them kits,
Prepare the application in early diagnosis of cancer or the reagent of targeting cancer therapy detection.
10. a kind of sequencing approach of small RNA, it is characterised in that：Including using the library construction described in claim 1 or 2
Method is carried out library construction, then constructed library is sequenced using CG microarray datasets.
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| CN110872609A (en) * | 2018-09-04 | 2020-03-10 | 深圳华大基因科技服务有限公司 | Method for accurately establishing library and sequencing small RNA molecules and application |
| CN111349718A (en) * | 2018-12-21 | 2020-06-30 | 深圳华大智造科技有限公司 | Primer group for pathogenic nucleic acid amplification, pathogenic nucleic acid detection library construction method and pathogenic detection method |
| CN112301130A (en) * | 2020-11-12 | 2021-02-02 | 苏州京脉生物科技有限公司 | Marker, kit and method for early detection of lung cancer |
| CN112840036A (en) * | 2018-08-15 | 2021-05-25 | 深圳华大生命科学研究院 | Gene chip and preparation method thereof |
| WO2023046163A1 (en) * | 2021-09-26 | 2023-03-30 | 杭州诺辉健康科技有限公司 | Method and kit for nucleic acid library construction and sequencing |
| CN110872609B (en) * | 2018-09-04 | 2024-06-04 | 深圳华大基因科技服务有限公司 | Method for accurately constructing library and sequencing small RNA molecules and application thereof |
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| CN112840036A (en) * | 2018-08-15 | 2021-05-25 | 深圳华大生命科学研究院 | Gene chip and preparation method thereof |
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| CN111349718A (en) * | 2018-12-21 | 2020-06-30 | 深圳华大智造科技有限公司 | Primer group for pathogenic nucleic acid amplification, pathogenic nucleic acid detection library construction method and pathogenic detection method |
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| WO2023046163A1 (en) * | 2021-09-26 | 2023-03-30 | 杭州诺辉健康科技有限公司 | Method and kit for nucleic acid library construction and sequencing |
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