CN109136372A - It is a kind of based on illumina platform breast cancer parting detection build library kit - Google Patents
It is a kind of based on illumina platform breast cancer parting detection build library kit Download PDFInfo
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- CN109136372A CN109136372A CN201810895524.XA CN201810895524A CN109136372A CN 109136372 A CN109136372 A CN 109136372A CN 201810895524 A CN201810895524 A CN 201810895524A CN 109136372 A CN109136372 A CN 109136372A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
Abstract
The invention belongs to field of biotechnology, and in particular to it is a kind of based on illumina platform breast cancer parting detection build library kit, including two box bodys and the clamshell box cover being connected with the box body, two box bodys are respectively A box body and B box body；The first paper support is placed in the A box body, and 20 reagent hole slots are set in the first paper support and/or centrifuge tube places loading slot；The second paper support is placed in B box body, is provided with 6 reagent hole slots in the second paper support.The features such as kit of the present invention has the breast cancer express spectra parting that can not only carry out flesh tissue but also can carry out the molecule parting of breast cancer FFPE sample, parting accuracy height, high sensitivity, stability is strong；The requirement of detection sensitivity can either be met, and a large amount of capital can be saved, in addition also have the characteristics that easy to operate, considerable economic benefit can not only be brought, moreover it is possible to realize quickly detection.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to it is a kind of based on illumina platform breast cancer parting detection build
Library kit.
Background technique
Breast cancer is in clinic still with patient age, primary tumor size, clinical stages, histological grade, armpit lymph at present
It carries down the indexs such as shifting and hormone receptor status, predicting prognosis of breast cancer and guidance selection therapeutic scheme.But breast cancer individual difference
Property is more significant, and above-mentioned standard can not further calculate to a nicety prognosis, it is difficult to meet breast cancer treatment regimen " individuation ".Compare
The gene expression profile for analyzing normal breast tissue and tumour, can specify the gene expression difference of mRNA level in-site, thus according to base
Because of the Clinical Outcome of expression prediction patient, corresponding treatment measure is taken.Various breast cancer labels based on gene expression profile
More objective, accurate prognosis prediction ability is shown, there is good development prospect.
From the appearance of intermediate gene chip the 1990s, just it is widely used in the relevant gene expression profile point of cancer
Analysis, and for predicting in human cancer that complicated and diversified biological characteristics are sought peace Clinical symptoms.Although gene expression spectrum analysis is wide
Application in the general research applied to human cancer, but in terms of the clinical treatment of patient is by great limitation.This be by
In most of clinical case tumor sample obtained at present be all formaldehyde fix or paraffin embedding by way of store, and very
It is few to be saved by way of cryopreservation.In order to attempt using these tumor samples abundant, researcher attempts to pass through RT-PCR
Or the modes such as DASL carry out the analysis of expression quantity, but these methods can not carry out accurately quantitatively, and can only be to minority
Known transcript is quantified.
In recent years, with the appearance of two generations sequencing and the continuous decline of sequencing cost, the side that can be sequenced by two generations
Formula analyzes the expression of mRNA in FFPE sample, to overcome limitation of the microarray technology for FFPE pattern detection, and right
Functional genomics generates revolutionary impact.
Traditional mRNA sequencing technologies are then to interrupt mRNA reverse transcription at cDNA by oligo dT reverse transcription primer
Build library or by the way that the mRNA for having oligo dA tail to be enriched with and then interrupt, reverse transcription, Jian Ku.The two methods all need
The RNA for obtaining high quality is achieved.
With the development of high throughput sequencing technologies, there is researcher to pass through the flesh tissue sample that will be matched and the fixed group of formaldehyde
It knits sample and carries out quantifying for tissue gene with the method for micro-array chip and the method for high-flux sequence respectively, as a result, it has been found that passing through
The gene expression dose correlation of flesh tissue and fixing organization that the method for high-flux sequence obtains is higher, and related coefficient reaches
0.82, and organize to obtain the related coefficient of result only 0.54, correlation to flesh tissue and FFPE by HEEBO micro-array chip
It is poor.
Major part clinical case laboratory tumor sample obtained is all to be fixed by formaldehyde or paraffin embedding at present
Mode stores, and is seldom saved by way of cryopreservation.In order to attempt using these tumor samples abundant, researcher is logical
The mode for crossing RT-PCR or DASL carries out the analysis of expression quantity, but these methods can not carry out accurately quantitatively, and can only
A small number of known transcripts is quantified, thus by this programme can be obtained from FFPE sample more reliable data come
Source.
Since breast cancer is a kind of disease of height heterogeneity, type and characteristic are also varied, and traditional
The effect in clinical application such as intrinsic parting, 21 gene risk profiles and 70 gene prognosis is limited, is sequenced and is obtained by two generations
Data information more wide and reliable data source can be provided for the molecule parting of breast cancer, may be breast cancer essence
Quasi- parting and treatment provide broader prospect.
Summary of the invention
It is mainly the transcription carried out with flesh tissue in view of the existing mRNA sequencing about breast cancer and quantitative means
Group sequencing, has higher requirement for sample, can not adapt to the needs of clinical diagnosis.The object of the present invention is to provide one kind to be not required to
The preferable mRNA of quality is wanted, even if the breast cancer parting based on illumina platform of degradation sample also available more excellent result
Library kit is built in detection.
In order to achieve the above-mentioned object of the invention, a kind of the technical solution adopted by the present invention are as follows: cream based on illumina platform
Library kit is built in the detection of gland cancer parting, it is characterised in that: including two box bodys and the clamshell box cover being connected with the box body,
Two box bodys are respectively A box body and B box body；The first paper support is placed in the A box body, 20 examinations of setting in the first paper support
Agent hole slot and/or centrifuge tube place loading slot, and 20 reagent hole slots and/or centrifuge tube, which are placed in loading slot, is respectively put into 1
Pipe Bio-B-TVN primer, 1 pipe dNTP mix, 1 pipe 5xFirst-strand buffer, 1 pipe 0.1M DTT, 1 pipe RNase
Inhibitor, 1 pipe Superscript, III reverse transcriptase, 1 pipe 10x TaqDNA polymerase Buffer, 1 pipe TaqDNA
Polymerase, 1 pipe RNase H, 1 pipe Terminal transferase buffer, 1 pipe ddNTP, 1 pipe 500mM EDTA are molten
Liquid, 1 pipe, 100 μM of Primer-A-Random primer solutions, 2 pipe EB, 3 pipe Phusion Master Mix, 1 pipe Primer-A draw
Object, 1 pipe Primer-B primer；The second paper support is placed in B box body, is provided with 6 reagent hole slots in the second paper support, is put respectively
Enter 1 bottle of nuclease-free water, 1 bottle of Ampure XP magnetic bead, 1 bottle of DyanabeadsMyone Streptavidin C1 magnetic bead, 1 bottle
0.1M NaOH, 2 bottles of Wash Buffer.
The Bio-B-TVN primer is the reverse transcriptase primer sequence of the anchor primer containing 19 oligoT and sequence measuring joints
Column.
3 ' the ends of Primer-A have the illumina joint sequence of 6 base random primers.
Bio-B-TVN primer, dNTP mix, 5xFirst-strand buffer, DTT, RNase inhibitor,
The pipe lid of III reverse transcriptase of Superscript has a blue gasket, Terminal transferase buffer,
DdNTP mix, terminal transferase, 500mM EDTA pipe lid have green gasket, the Primer-A-
Random primer solution, TaqDNA polymerase, 10x TaqDNApolymerase Buffer, Rnase H pipe lid have
Red gasket, Phusion Master Mix2X, primer-A and Primer B primer pipe lid have yellow gasket, institute
The EB and nuclease-free water stated has transparent pipe lid；The Ampurexp magnetic bead, DyanabeadsMyone Streptavidin
C1 magnetic bead, NaOH and Wash Buffer pipe lid have white gasket.
The Bio-B-TVN primer is as shown in SEQ ID No.1；Primer-A-Random primer such as SEQ ID No.2 institute
Show；Primer-A primer is as shown in SEQ ID No.3；Any one sequence institute of Primer-B primer such as SEQ ID No.4-6
Show.
The concrete composition of library kit is built in the breast cancer parting detection are as follows:
Compared with the existing technology, the invention has the benefit that kit of the present invention is fresh with can both carry out
The breast cancer express spectra parting of tissue, the molecule parting that can carry out breast cancer FFPE sample again, parting accuracy are high, sensitivity
The features such as height, stability is strong；The requirement of detection sensitivity can either be met, and a large amount of capital can be saved, in addition also there is behaviour
Make the features such as simple, considerable economic benefit can not only be brought, moreover it is possible to realize quickly detection.
Detailed description of the invention
The following drawings forms a part of present specification, and for further illustrating certain aspects of the invention.This hair
It is bright to be come by reference to one or more of these attached drawings in conjunction with the detailed description of specific embodiment proposed in this paper
It more fully understands.
Fig. 1 is the structural representation that library kit A is built in the breast cancer parting detection of the present invention based on illumina platform
Figure；
Fig. 2 is the knot for the paper support that library kit A is built in the breast cancer parting detection of the present invention based on illumina platform
Structure schematic diagram；
Fig. 3 is the structural representation that library kit B is built in the breast cancer parting detection of the present invention based on illumina platform
Figure；
Fig. 4 is the knot for the paper support that library kit B is built in the breast cancer parting detection of the present invention based on illumina platform
Structure schematic diagram；
Wherein: 1, A box box body；2, A box box cover；3, A box box paper support；4, B box box body；5, B box box cover；6, B box paper support；
11, Bio-B-TVN primer；12,dNTP mix；13,5xFirst-strand buffer；14,DTT(0.1M)；15,RNase
inhibitor；16, III reverse transcriptase of Superscript；17,10x TaqDNA polymerase Buffer；18,TaqDNA
polymerase；19,RNase H；20,Terminal transferase buffer；21,ddNTP；22,500mM EDTA is molten
Liquid；23, Primer-A-Random primer solution (100 μM)；24,EB；25,EBPhusion Master Mix；26,Primer-
A primer；27, Primer-B primer；28.Phusion Master Mix；29.Phusion Master Mix；30,Phusion
Master Mix；31,Wash Buffer；32, Ampure XP magnetic bead；33,0.1M NaOH；34, nuclease-free water；35,
DyanabeadsMyone Streptavidin C1 magnetic bead.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
It should be appreciated that such as " having " used herein, " comprising ", " ", "comprising" term are not precluded one
Or the presence or addition of a number of other elements or combinations thereof.
The invention belongs to biological diagnosis detection field, it is related to being sequenced in a manner of high-throughput and is expressed in breast cancer FFPE sample
Multiple genes.More specifically, this disclosure relates to a kind of library construction of the sequencing for 3 ' end of mRNA sample of degrading with
And sequencing analysis, to parting and and risk assessment are carried out in breast cancer tissue with the express spectra difference of breast cancer related gene
Method.Wherein detection method includes the extraction of total serum IgE in (1) tumor patient tissue FFPE sample.(2) pass through reverse transcriptase and draw
Object reverse transcription RNA is then the cDNA with polyA tail is passed through by the cDNA of the affine magnetic bead combination biotin labeling of streptomysin
Taq enzyme synthesize two chain cDNA. (3) of belt lacing by the PrimerB primer with barcode and primerA primer pair library into
Row amplification.(4) upper machine sequencing is carried out to amplification library.(5) reads mapping, gene expression amount point are carried out to sequencing data
Analysis and mastocarcinoma gene expression pattern analysis and Genotyping.
Temperature is higher due to the effect of denaturing formaldehyde and in embedding process for FFPE sample, it is easy to cause nucleic acid molecules
Fragmentation and chemical modification, to be difficult to therefrom extract complete RNA.
Since there are the heterogeneities of height for breast cancer cell, the treatment of patient with breast cancer is used for by traditional classifying method
In, even if its therapeutic effect of same kind still has very big difference, and breast cancer parting and prognosis are carried out by express spectra
Risk assessment is accurate breast cancer accurately typing, provides possibility.Each primer sequence of table 1
| Title | Sequence |
| Bio-B-TVN | Biotin-5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT TTTTTTTTTTTTTTTTTTTVN-3′ |
| Primer-A-Random | 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNN-3′ |
| Primer A | 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ |
| Primer B1 | 5′-CAAGCAGAAGACGGCATACGAG-ATCACGTT-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ |
| Primer B2 | 5′-CAAGCAGAAGACGGCATACGAG-CGATGTTT-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ |
| Primer B3 | 5′-CAAGCAGAAGACGGCATACGAG-TTAGGCAT-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ |
On the one hand, present embodiment in some terms, FFPE sample is extracted through dimethylbenzene and RNeasy FFPE Kit.
Since there are polyA stern constructions can guarantee the relatively stable of 3 ' end sequences for the end mRNA3 ', pass through this
Structure can effectively filter out the 3 ' ends of mRNA from total serum IgE.
There are biotin labeling in the 5 ' ends of the Bio-B-TVN of this kit, can be with the affine magnetic bead covalent bond of streptomysin
To which the mRNA with polyA tail be screened.
5 ' the ends of preferred chain synthesis reverse transcriptase primer Bio-B-TVN are with biotin labeling and illumina
P7 sequencing primer sequence is identical；3 ' the ends of reverse transcriptase primer Bio-B-TVN are the oligo primer with 19 T, with mRNA3 '
The oligoA at end can carry out complementary pairing, and the V of 3 ' ends represents base A or C or G, and N represents base A or T or C or G.
The cDNA covalent bond synthesized by strepavidin magnetic beads with a chain before carrying out the synthesis of two chains, then by preferred
Two chain synthetic primers carry out two chain synthesis, while specificity is identical with illumina sequencing primer in the addition of the end mRNA5 '
Two chain synthetic primers, 3 ' ends are the random sequence of 7 bases.
Preferably by the not Primer A primer of provided with bar code (barcode) and with 8 bases after the synthesis of library
Mark (index) Primer B primer pair library expanded, library both ends add P5, P7 connector, the joint sequence with
The complementary combination of P5, P7 oligonucleotide chain (oligo) on flow cell (flowcell).It can pass through after the sequencing of upper machine simultaneously
Index distinguishes different samples.
Sample can directly be derived from subject or available from third party.Sample can be the cancer of patient with breast cancer's FFPE sample
Position.
The composition of this kit most preferably includes component shown in table 2:
The composition of 2 kit of table
The present invention refers to the method that RNeasy FFPE Kit (Kai Jie) tumour FFPE sample rna extracts comprising following step
It is rapid:
A. the tissue 2-3 piece for cutting away outermost exposure air first, then cuts 10 μ m-thicks, area 250mm2Tissue
2.
B. 1ml dimethylbenzene is added, abandons supernatant after mixing centrifugation, 150 μ l buffer-PDK are then added, is vortexed and mixes.
C. 150 μ l bufferPKD are added, 10 μ l Proteinase Ks are then added, is incubated for 15min at 56 DEG C, is incubated at 80 DEG C
15min。
D. colourless lower phase is transferred in new 2ml centrifuge tube, after being incubated for 3min on ice, 20000xg is centrifuged 15min.On
It is transferred to clearly in new centrifuge tube, not encounter sediment.
E. the DNase Booster Buffer (about 16-25 μ l) and 10 μ l DNase I of 1/10 sample volume is added
stock solution.Liquid is mixed, wink is from incubation at room temperature 15min.
F. 320 μ l Buffer RBC are added, thoroughly mix.The mixing of 720 μ l dehydrated alcohols is added, transfer sample is extremely
On RNeasy MinElute spin column, 8000xg is centrifuged 15s, abandons liquid.
G. it being added in 500 μ l Buffer RPE to RNeasy MinElute spin column, 8000xg is centrifuged 2min,
Abandon collecting pipe.
H. RNeasy MinElute spin column is placed back in a new collecting pipe, uncap centrifugation 5min at full speed.
I. collecting pipe is abandoned, RNeasy MinElute spin column is put into new 1.5ml collecting pipe, in spin
14-30 μ l is added without RNA enzyme water (RNase-free water) in column film center, is centrifuged 1min at full speed, collects eluent.
The total serum IgE of acquisition is purified, library construction, upper machine sequencing and data analysis comprising following steps:
A. 11 μ l total serum IgE 1-3 μ g after extracting are taken, the Bio-B-TVN primer of 50 μM of 1 μ l, Bio-B-TVN primer is added
5 ' ends are consistent with the P7 primer on illuminaflowcell, and wherein V represents (A, G or C, N represent randomized bases), add 1 μ
The dNTP (10mM) of l.In 65 DEG C of incubation 5min, it is subsequently placed at least 1min on ice, 4 μ l 5xFirst-strand are added
Buffer, 1 μ l 0.1M DTT, 1 μ l RNase inhibitor (RNase inhibitor) (invetrogen), 1 μ l
Superscript IIIreverse transcriptase reverse transcriptase (200U/ μ l；Invitrogen it) is incubated at 50 DEG C
60min carries out reverse transcription reaction, and 80 μ l water, 70 DEG C of 15min inactivations are then added.
B. magnetic bead is purified by 1.5X Ampure and is incubated at room temperature 5min, be placed on magnetic frame and stand 5min, clarified to solution
After abandon supernatant, the 80% ethyl alcohol rinsings of the 200 fresh configurations of μ l is added twice, and washed with 50 μ l EB (10mM Tris-Hcl, PH8)
It is de-, see Fig. 2.
C.50 15 μ l 10X terminal transferase buffer (NEB cat# are added in μ l eluted product
M0315S), the end terminaltransferase of the sterile water of 130 μM of ddNTP mix, 80 μ l of 3ul and 2ul are shifted
Enzyme (NEB cat#M0315S) is in 37 DEG C of incubation 1h.Then the EDTA solution of 6.25 μ l 500mM is added and terminates to final concentration 20mM
Reaction.
D. Streptavidin MagneSphere (the DyanabeadsMyOne Streptavidin of 5 μ l is added in the product of 156 μ l
C1, Cat#65001), it is incubated at room temperature 20min.Then 100 μ l NaOH (0.1M) piping and druming is added to mix, is incubated at room temperature 5min.It sets
In abandoning supernatant after solution clarification on magnetic frame, cleaned twice with nuclease-free water.
E. Primer-A-Random (100 μM) primer of 1 μ l is added, adds the TaqDNA of 5 μ l 10X
Polymerase buffer (NEB), 1 μ l 10mM dNTP, adds water to supply 49 μ l.The Taq DNA polymerase of 1 μ l is added
In 25 DEG C of incubation 1h, 72 DEG C of extension 30s, 75 DEG C of 5min are kept polymerase (5 μ l, NEB).It is dense to end that 1 μ l 500mM EDTA is added
10mM is spent, reaction is terminated.It is placed on magnetic frame and abandons supernatant after solution clarification, with 150 μ l wash buffer (10mMTris-
HCL, PH 7.5,1mMEDTA and 0.1%Tween-80 or Triton-X100) it cleans twice, it is then resuspended in 24 μ l water.
F. 25 μ l Phusion Master Mix (High Fidelity Enzyme Mix) 2X (NEB) and 0.5 μ l universal primer is added
Primer-B (10 μM) primer and 0.5 μ l primer A (10 μM) primer are expanded, and amplification condition is 96 DEG C of 30s, and 18 are followed
Ring (96 DEG C of 10s, 65 DEG C of 10s, 72 DEG C of 10s), 72 DEG C of 5min.PCR product 1.5x Ampure XP magnetic beads for purifying, and with 20 μ l
EB elution.Primer-B is the one or more of Primer B1, B2, B3.
G. library passes through VAHTS Library Quantification Kit for Illumina after eluting (promise is only praised)
Quantification kit and 5 fluorescence quantitative PCR instrument of IQ are quantified.
H. final library is sequenced according to the data volume of 3G by nextseq 500.
As shown in Figs 1-4, library kit is built in the breast cancer parting detection that the present invention discloses a kind of illumina platform, is wrapped
It includes: including two box bodys and the clamshell box cover being connected with the box body, placing a paper support, the paper support in the A box body
20 reagent hole slots of upper setting and centrifuge tube place loading slot, are respectively put into 1 pipe Bio-B-TVN primer, 1 in 20 hole slots
Pipe dNTP mix, 1 pipe 5xFirst-strand buffer, 1 pipe DTT (0.1M), 1 pipe RNase inhibitor, 1 pipe
III reverse transcriptase of Superscript, 1 pipe 10x TaqDNA polymerase Buffer, 1 pipe TaqDNA polymerase, 1
Pipe Terminal transferase buffer, 1 pipe ddNTP, 1 pipe 500mM EDTA solution, 1 pipe Primer-A-Random draw
Object solution (100 μM), 2 pipe EB, 1 pipe Primer-A primer, 1 pipe Primer-B primer, 3 pipe Phusion Master Mix.B box
A paper support is placed in vivo, is provided with 6 reagent hole slots in affiliated paper support, is respectively put into 1 bottle of nuclease-free water, 1 bottle of Ampure XP
Magnetic bead, 1 bottle of DyanabeadsMyone Streptavidin C1 magnetic bead, 1 bottle of 0.1M NaOH, 2 bottles of Wash Buffer.
Specific operation process: reverse transcription is carried out from OligoA tail end to the fragmentation mRNA obtained in FFPE sample, then
2 chain synthesis are carried out by random primer, PCR amplification is carried out later and obtains the sequencing library with oligoA, at another to FFPE
The fragmentation mRNA obtained in sample carries out reverse transcription from OligoA tail end, then carries out 2 chain synthesis by random primer, later
It carries out PCR amplification and obtains the sequencing library with oligoA.
Kit of the present invention has the breast cancer express spectra parting that can not only carry out flesh tissue but also can carry out mammary gland
The features such as molecule parting of cancer FFPE sample, parting accuracy are high, high sensitivity, stability is strong；It is sensitive that detection can either be met
The requirement of degree, and can save a large amount of capital, in addition also has the characteristics that easy to operate, can not only bring considerable economic benefit,
Also it is able to achieve quick detection.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table
<110>the biomedical scientific and technological Nanjing Co., Ltd of Jiangsu Su Bo
<120>library kit is built in a kind of breast cancer parting detection based on illumina platform
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<141> 2018-08-08
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Claims (6)
1. library kit is built in a kind of breast cancer parting detection based on illumina platform, it is characterised in that: including two box bodys
And the clamshell box cover being connected with the box body, two box bodys are respectively A box body and B box body；The is placed in the A box body
One paper support, 20 reagent hole slots are arranged in the first paper support and/or centrifuge tube places loading slot, 20 reagent hole slots
And/or centrifuge tube is placed in loading slot and is respectively put into 1 pipe Bio-B-TVN primer, 1 pipe dNTP mix, 1 pipe 5xFirst-strand
Buffer, 1 pipe 0.1M DTT, 1 pipe RNase inhibitor, 1 pipe Superscript, III reverse transcriptase, 1 pipe 10x TaqDNA
Polymerase Buffer, 1 pipe TaqDNA polymerase, 1 pipe RNase H, 1 pipe Terminal transferase
Buffer, 1 pipe ddNTP, 1 pipe 500mM EDTA solution, 1 pipe, 100 μM of Primer-A-Random primer solutions, 2 pipe EB, 3 pipes
Phusion Master Mix, 1 pipe Primer-A primer, 1 pipe Primer-B primer；Place the second paper support in B box body, described the
It is provided with 6 reagent hole slots in two paper supports, is respectively put into 1 bottle of nuclease-free water, 1 bottle of Ampure XP magnetic bead, 1 bottle
DyanabeadsMyone Streptavidin C1 magnetic bead, 1 bottle of 0.1M NaOH, 2 bottles of Wash Buffer.
2. library kit is built in a kind of breast cancer parting detection based on illumina platform according to claim 1, special
Sign is: the Bio-B-TVN primer is the reverse transcriptase primer sequence of the anchor primer containing 19 oligoT and sequence measuring joints.
3. library kit is built in a kind of breast cancer parting detection based on illumina platform according to claim 1, special
Sign is: the 3 ' ends of Primer-A have the illumina joint sequence of 6 base random primers.
4. library kit is built in a kind of breast cancer parting detection based on illumina platform according to claim 1, special
Sign is, Bio-B-TVN primer, dNTP mix, 5xFirst-strand buffer, DTT, RNase inhibitor,
The pipe lid of III reverse transcriptase of Superscript has a blue gasket, Terminal transferase buffer,
DdNTP mix, terminal transferase, 500mM EDTA pipe lid have green gasket, the Primer-A-
Random primer solution, TaqDNA polymerase, 10x TaqDNA polymerase Buffer, Rnase H pipe lid have
Red gasket, Phusion Master Mix2X, primer-A and Primer B primer pipe lid have yellow gasket, institute
The EB and nuclease-free water stated has transparent pipe lid；The Ampurexp magnetic bead, DyanabeadsMyone Streptavidin
C1 magnetic bead, NaOH and Wash Buffer pipe lid have white gasket.
5. library kit is built in a kind of breast cancer parting detection based on illumina platform according to claim 1, special
Sign is that the Bio-B-TVN primer is as shown in SEQ ID No.1；Primer-A-Random primer such as SEQ ID No.2 institute
Show；Primer-A primer is as shown in SEQ ID No.3；Any one sequence institute of Primer-B primer such as SEQ ID No.4-6
Show.
6. library kit is built in a kind of breast cancer parting detection based on illumina platform according to claim 1, special
Sign is that the kit specifically contains following component:
。
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Application publication date: 20190104 |