CN109207557A - A kind of low initial amount banking process suitable for next-generation microarray dataset - Google Patents
A kind of low initial amount banking process suitable for next-generation microarray dataset Download PDFInfo
- Publication number
- CN109207557A CN109207557A CN201710551477.2A CN201710551477A CN109207557A CN 109207557 A CN109207557 A CN 109207557A CN 201710551477 A CN201710551477 A CN 201710551477A CN 109207557 A CN109207557 A CN 109207557A
- Authority
- CN
- China
- Prior art keywords
- dna
- product
- magnetic bead
- pcr amplification
- purifying
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Computational Biology (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of low initial amount banking process suitable for next-generation microarray dataset, comprising: DNA fragmentation；It repairs end；Product is repaired with magnetic beads for purifying end, and retains magnetic bead in system；Connector connection；Twice with magnetic beads for purifying connector connection product, and magnetic bead is removed；Using the connector connection product of purifying as template, PCR amplification is carried out；First time pcr amplification product is screened with magnetic bead；The product that segment is screened is hybridized with chip；Hybrid product is captured with magnetic bead, is then gone by elution unless capturing target gene and other impurity；PCR amplification is carried out as template using the DNA with magnetic bead after eluting, target gene signal is further amplified；Twice with second of pcr amplification product of magnetic beads for purifying.Method of the invention can detect tetra- kinds of SNV, Indel, fusion and CNV mutation type samples simultaneously, build library initial amount and be reduced to 100ng hereinafter, can satisfy most detections for puncturing sample.
Description
Technical field
The present invention relates to sequencing technologies fields, and in particular to a kind of low initial amount suitable for next-generation microarray dataset builds library
Method.
Background technique
Aspiration biopsy is the main method that Bone and soft tissue tumors obtain histopathogenic diagnosis, is worn by special puncture needle
Thorn or suction biopsy lymph node, marrow, liver, spleen, kidney etc. extract tissue specimen.Examine the correlation for puncturing sample
The prior art mainly has: library is built in Ion Ampliseq technology and conventional capture.
Ion Ampliseq technology, obtains target gene fragment by multiplex PCR, then carries out connector connection, pre--PCR
(pre-PCR) operation such as.Ion Ampliseq technology can be divided into two kinds of Ampliseq DNA and Ampliseq RNA, Ampliseq
DNA is mainly for single nucleotide variations (SNV) and inserts and delete (Indel) two kinds of mutation types；The main needle of Ampliseq RNA
To fusion (Fusion) mutation type.Ion Ampliseq technology can will build the reduction of library initial amount by superelevation multiplexed PCR amplification
To 10ng, but this method needs to extract DNA and RNA respectively, then build respectively library could by SNV, Indel of sample and
Tri- kinds of mutation type pattern detections of Fusion are complete.
Library is built in conventional capture, by carrying out fragmentation to DNA and purifying, then carries out end reparation and purifying, connector connect
It connects and purifies ,-PCR (pre-PCR) and purifying, hybridization and elution, rear-PCR (post-PCR) and purify in advance.Routine is caught
SNV, Indel, Fusion and four kinds of mutation type samples of gene copy number variation (CNV) can be detected simultaneously by obtaining database technology, but
It is high to build library initial amount requirement, because needing to be purified for every single step reaction in conventional capture database technology, one step of every increase
Purifying will lose a large amount of DNA information, need very high build so as to cause traditional banking process for tumor tissues
Library initial amount.Small sample (as punctured sample) accounts for very at high proportion in clinical tumor tissue samples, but small sample is due to tissue mass
It is few, it is difficult therefrom to obtain the DNA higher than 500ng, therefore conventional capture database technology can not be to puncture pattern detection.
Summary of the invention
The present invention provides a kind of low initial amount banking process suitable for next-generation microarray dataset, can detect simultaneously SNV,
Tetra- kinds of mutation type samples of Indel, Fusion and CNV build library initial amount and are reduced to 100ng hereinafter, can satisfy the overwhelming majority
Puncture the detection of sample.
A kind of low initial amount suitable for next-generation microarray dataset is provided according to an embodiment of the present and builds library side
Method includes the following steps:
(1) DNA of extraction DNA fragmentation: is broken into segment；
(2) end is repaired: the DNA fragmentation interrupted being added to end and is repaired in reaction solution, is incubated for；
(3) it purifies for the first time: repairing product with magnetic beads for purifying end, and retain magnetic bead in system；
(4) connector connects: product being repaired in end after purification and is added in connection reaction solution together with magnetic bead, simultaneously
Connector is added, is incubated for；
(5) it purifies for second: twice with magnetic beads for purifying connector connection product, and removing magnetic bead；
(6) first time PCR amplification: using the connector connection product of purifying as template, PCR amplification is carried out；
(7) segment is screened: screening first time pcr amplification product with magnetic bead；
(8) hybridize: the first time pcr amplification product that segment is screened is hybridized with chip；
(9) it captures and elutes: hybrid product being captured with magnetic bead, then gone by elution unless capturing target gene
With other impurity；
(10) second of PCR amplification: PCR amplification is carried out as template using the DNA with magnetic bead after eluting, to be further amplified
Target gene signal；
(11) third time purifies: twice with second of pcr amplification product of magnetic beads for purifying.
Further, DNA derives from tumor puncture sample in above-mentioned steps (1).
Further, the amount of DNA is 10ng to 100ng in above-mentioned steps (1).
Further, DNA is broken into the segment that size is 200bp or so in above-mentioned steps (1).
Further, product is repaired with 1.8 times of XP magnetic beads for purifying ends AMPure in above-mentioned steps (3).
Further, with 1.2 times of AMPure XP magnetic beads for purifying connector connection products in above-mentioned steps (5).
Further, expanded respectively with 0.8 times and 0.2 times of AMPure XP magnetic bead screening first time PCR in above-mentioned steps (7)
Increase production object.
Further, before first time pcr amplification product is hybridized with chip in above-mentioned steps (8), by every chip
The DNA total amount for hybridizing 800ng calculates, and 4-8 sample is mixed into a library in proportion.
Further, hybrid product is captured with Streptavidin MagneSphere in above-mentioned steps (9).
Further, with 1.5 times of AMPure XP second of pcr amplification products of magnetic beads for purifying in above-mentioned steps (11).
Banking process of the invention is the detection method that library foundation is built based on capture.Compared to other banking process, have
Following advantage:
(1) low initial amount, for the library construction of traditional hybrid capture, the present invention passes through to the excellent of technical system
Change, so that entire library construction system can be suitably used for the clinical detection of the puncture sample of small sample amount (≤100ng)；Specifically,
Technology optimization includes: that end reparation is directly carried out after (a) DNA is interrupted, after AMPure XP magnetic beads for purifying, by band
Magnetic bead DNA directly carry out connector connection reaction, both can guarantee connection reaction efficiency, be also avoided that purifying caused by DNA
Loss.(b) during connector connects, by 3 ' the single-stranded polishings in end of connector at double-strand, product then is repaired with end and is carried out
Blunt end cloning can shorten the PCR reaction time in this way, improve PCR amplification efficiency.
(2) variation type detection type is more, using chip capture technique, can detect simultaneously SNV, Indel, Fusion and
Tetra- kinds of mutation types of CNV.
(3) high sensitivity, relative to Ion Ampliseq technology, the detection frequency of mutation of the present invention in SNV, InDel can
Down to 1%, the detection frequency of mutation of Fusion, CNV can be down to 10%.
(4) application is wide, by hybridizing different chips, is applicable to the detection of various tumor tissues samples.
(5) it is short to build the library time, and the present invention captures Library development flow relative to conventional, and step is more succinct, and it is shorter to build the library time.
Detailed description of the invention
Fig. 1 is the banking process flow chart of one embodiment of the present of invention；
2100 quantitative result of Agilent in Fig. 2 embodiment of the present invention.
Specific embodiment
Below by specific embodiment combination attached drawing, invention is further described in detail.In the following embodiments and the accompanying drawings
In, many datail descriptions are in order to enable the present invention can be better understood.However, those skilled in the art can be without lifting an eyebrow
Recognize, part of feature is dispensed in varied situations, or can be substituted by other materials, method.
In some cases, the relevant some operations of the present invention there is no display in the description or describe, this is in order to avoid this
The core of invention is flooded by excessive description, and to those skilled in the art, these correlations behaviour is described in detail
Be not necessary, they according to the general technology knowledge of description and this field in specification can be complete decorrelation
Operation.
As shown in Figure 1, in an embodiment of the present invention, the low initial amount suitable for next-generation microarray dataset builds library side
Method includes the following steps:
(1) DNA is extracted: for example, using QIAGEN company QIAamp DNA FFPE Tissue Kit kit from Fu Er
DNA is extracted in fixed paraffin embedding (Formalin-Fixed and Parrffin-Embedded, the FFPE) sample of Malin.DNA
Tumor puncture sample can be derived from.
(2) DNA of extraction DNA fragmentation: is broken into segment.For example, 10ng to 100ng DNA is taken to be diluted to 50 μ L bodies
Product, it is 200bp or so (for example, 100-300bp, 150-250bp, 180- that DNA, which is then broken into size, using Ultrasonic Cell Disruptor
220bp or 190-210bp) segment.
(3) end is repaired: the DNA fragmentation interrupted being added to end and is repaired in reaction solution, is incubated for, for example, 20
DEG C be incubated for 30min.
(4) it purifies for the first time: it is primary with magnetic bead (for example, 1.8 times of AMPure XP magnetic beads) purifying end reparation product, and
Retain magnetic bead in system.
(5) connector connects: product being repaired in end after purification and is added in connection reaction solution together with magnetic bead, simultaneously
It is added connector (for example, A connector and P connector), after piping and druming mixes, is incubated for, for example, in 20 DEG C of incubation 30min.
(6) it purifies for second: twice with magnetic bead (for example, 1.2 times of AMPure XP magnetic beads) purifying connector connection product, and
Remove magnetic bead.
(7) first time PCR (Pre-PCR) is expanded: using the connector connection product of purifying as template, carrying out PCR amplification.
(8) segment is screened: screening first time PCR with magnetic bead (for example, respectively with 0.8 times and 0.2 times of AMPure XP magnetic bead)
Amplified production.
(9) hybridize: the first time pcr amplification product that segment is screened is hybridized with chip.For example, miscellaneous by every chip
It hands over 800ng DNA total amount to calculate, 4-8 sample is mixed to (pooling) into a library in proportion, then carried out with chip miscellaneous
It hands over.
(10) it captures and elutes: hybrid product being captured with magnetic bead (for example, M-280 Streptavidin MagneSphere), so
It is gone afterwards by elution unless capturing target gene and other impurity.
(11) second of PCR (Post-PCR) amplification: carrying out PCR amplification as template using the DNA with magnetic bead after eluting, with
Target gene signal is further amplified.
(12) third time purifies: purifying second of pcr amplification product two with magnetic bead (for example, 1.5 times of AMPure XP magnetic beads)
It is secondary.
(13) library Quality Control: for example, carrying out nucleic acid quantification and segment to library with qubit/qPCR and Agilent 2100
Size Quality Control.
(14) machine is sequenced on: carrying out upper machine sequencing to the library of Quality Control qualification.
Below by way of the specific embodiment technical solution that the present invention will be described in detail, it should be understood that embodiment is only exemplary
, it should not be understood as limiting the scope of the invention.
Embodiment one
The FFPE sample for choosing 10 upper patients with lung adenocarcinoma extracts total first with FFPE sample DNA extracts kit
DNA, then library constructing method through the invention constructs library.
1. library construction
1.1DNA fragmentation: the DNA of 100ng is diluted to 50 μ with the water (Nuclease-free water) of nuclease free
L after mixing, is interrupted DNA to the segment of 200bp or so with Ultrasonic Cell Disruptor.
Sample such as is interrupted using Covaris S220, it is as follows to interrupt condition: Peak Incident Power (w): 175；
Duty Factor:10%；Treatment Time (s): 170.
Instrument such as is interrupted using Bioruptor, then is carried out as follows: opening 30s, closes 30s, 6 switch cycles；Repetition is beaten
It is 3 times disconnected.
It is to be noted that having interrupted every time, sample is taken out from instrument, vortex oscillation 10s mixes wink from ice bath
3min is interrupted next time again.
The sample interrupted is transferred in clean EP pipe.
Repair 1.2 ends: the fragmentation DNA after interrupting is added to end and repairs in reaction solution, is configured to shown in table 1
It prepares end and repairs reaction system, in 20 DEG C of incubation 30min, then repair product with 1.8 times of XP magnetic beads for purifying ends AMPure
Once, last eluted dna, does not remove magnetic bead.
Table 1
The connection of 1.3 connectors: connection reaction solution and connector (A connector and P connector) are added in DNA solution after purification, matches
It is set to coupled reaction system shown in table 2, in 20 DEG C of incubation 30min.
Table 2
A-X- connector and P- connector, particular sequence such as the following table 3 (the wherein thio-modification that * indicates front base):
Table 3
The purifying of 1.4 connector connection products: with 1.2 times AMPure XP magnetic beads for purifying connector connection product 1 time, eluted dna
When do not remove magnetic bead, 20%PEG solution repurity connection product 1 time of 1.2 times is then added.It specifically includes:
1.4.1 30min is balanced at room temperature using preceding be placed in PEG solution；
1.4.2 by after the EP pipe brief centrifugation equipped with the DNA after the connection of 50 μ L connectors, the PEG that 1.2 times of volumes are added is molten
Liquid blows and beats 10 mixings with pipettor；
1.4.3 standing 6min at room temperature combines magnetic bead sufficiently with DNA, then brief centrifugation 3s；
1.4.4 EP pipe magnetic frame is put into clarify up to liquid；
1.4.5 supernatant (being careful not to be drawn onto magnetic bead) is carefully removed with pipettor；
1.4.6 keep EP pipe on magnetic frame, the ethyl alcohol of 400 μ L 80% of addition, two weeks washing magnetic bead surfaces of tube, with
Remove salt ion and unadsorbed DNA etc.；
1.4.7 it stands, removes ethyl alcohol after solution clarification；
1.4.8 step 1.4.6 is repeated to step 1.4.7；
1.4.9 upper magnetic frame 1min, completely removes ethyl alcohol, and magnetic bead is uncapped and is placed in magnetic frame as far as possible after being slightly centrifuged
On, until magnetic bead surfaces are lackluster；
1.4.10 50 μ L elution buffers are added, magnetic bead is gently rinsed to from tube wall and blown and beaten 10 mixings, is entered
Next step.
1.4.11 step 1.4.1 is repeated to step 1.4.10；
1.4.12 10min is stored at room temperature so that DNA is eluted completely from magnetic bead；
1.4.13 EP pipe is put into magnetic frame to clarify up to liquid, the 33 μ L DNA eluted is transferred to a new EP
Guan Zhong.
1.5Pre-PCR: using the connection product of purifying as template, PCR reaction system as shown in table 4 below, amplification 12 are configured
A circulation, PCR program are as follows: 98 DEG C of 45s；(98 DEG C of 15s, 65 DEG C of 30s, 72 DEG C of 30s) 11 circulations；72 DEG C of 5min, 4 DEG C of ∞.
Table 4
A primer and the particular sequence of P1 primer are as shown in table 5 below:
Table 5
The screening of 1.6 segments: first with 0.8 times of AMPure XP magnetic beads for purifying PCR product, going magnetic bead (magnetic bead absorption large fragment),
Stay supernatant；Then add 0.2 times of AMPure XP magnetic bead (5 times of volume concentrations) into supernatant.Specific steps include:
1.6.1 30min is balanced at room temperature using preceding be placed in magnetic bead；
1.6.2 the DNA after 100 μ L Pre-PCR reaction is transferred in the EP pipe of 1.5mL, the magnetic bead of 0.8 times of volume is added,
10 mixings are blown and beaten with pipettor；
1.6.3 6min is stood at room temperature, combines magnetic bead sufficiently with DNA, then brief centrifugation 3 seconds；
1.6.4 EP pipe magnetic frame is put into clarify up to liquid；
1.6.5 supernatant is carefully shifted with pipettor (to be careful not to be drawn onto magnetic bead) into the EP pipe of new 1.5mL；
1.6.6 0.2 times of magnetic bead (5 times of concentration) is added in the EP pipe into step 1.6.5 equipped with supernatant, is blown and beaten with pipettor
10 mixings；
1.6.7 standing 8min at room temperature combines magnetic bead sufficiently with DNA, then brief centrifugation 3 seconds；
1.6.8 EP pipe magnetic frame is put into clarify up to liquid；
1.6.9 keep EP pipe on magnetic frame, the ethyl alcohol of 400 μ L 80% of addition, two weeks washing magnetic bead surfaces of tube, with
Remove salt ion and unadsorbed DNA etc.；
1.6.10 it stands, removes ethyl alcohol after solution clarification；
1.6.11 step 1.6.9 is repeated to step 1.6.10；
1.6.12 upper magnetic frame 1min, completely removes ethyl alcohol, and magnetic bead is uncapped and is placed in magnetic frame as far as possible after being slightly centrifuged
On, until magnetic bead surfaces are lackluster；
1.6.13 the water of 33 μ L nuclease frees is added, gently rinse magnetic bead from tube wall and blows and beats 10 mixings；
1.6.14 10min is stored at room temperature so that DNA is eluted from magnetic bead completely；
1.6.15 EP pipe is put into magnetic frame to clarify up to liquid, the 30 μ L DNA eluted is transferred to a new EP
Guan Zhong；
1.6.16 it is quantitative DNA to be carried out to Pre-PCR product using Qubit HS.
1.7 hybridization: (chip is synthesized every chip by the design of Aglient company, substantially has biotin labeling
Rna probe collection, the chip cover ALK, BRAF, EGFR, KRAS, NRAS, PIK3CA, DDR2, ERBB2, RET, ROS1, MET etc.
Lung cancer related gene hot spot mutation) corresponding DNA hybridization total amount be 800ng, respectively by the Pre-PCR purified product of every 5 samples
It is mixed into a library in 1:1 ratio, in corresponding hybridization reaction solution, 65 DEG C are incubated for for 24 hours.Specifically comprise the following steps:
1.7.1 it is need to be scaled for the sample of different sequence labels to be mixed into a library according to data, total amount is
800ng, concussion mix, brief centrifugation；
1.7.2 pipe lid is opened, nozzle is closed with sealed membrane, clean 10 μ L pipette tips is taken to stab several duck eyes on sealed membrane,
It is placed in vacuum concentration instrument to rotate and be evaporated, temperature setting is 45 DEG C；
1.7.3 the sample library system of enrichment target fragment is formulated for according to table 6 in centrifuge tube:
Table 6
Note: Ion Xpress_A_X block (500 μM) takes corresponding A_ according to the sequence label of mixing sample when mixing
Each 0.5 μ L mixing of block, adds the A_block mixed liquor of 0.6 μ L.
Reaction system is all transferred in a new PCR pipe, carries out prehybridization according to 7 reaction condition of table:
Table 7
1.7.4 according to 8 preparing hybrid buffer reaction system of table in a new 1.5ml centrifuge tube:
Table 8
It according to example reaction number, is added separately in PCR pipe by the amount of 13 μ L of each reaction, covers pipe lid, then will
It is put into PCR instrument, 65 DEG C of incubation at least 2min, does not have crystal settling that could use in confirmation system.
1.7.5 oligonucleotide library mixture (Oligo Library is prepared according to table 9 in a new PCR pipe
Mix):
Table 9
Reaction system is put into 65 DEG C of incubation 2min in PCR instrument.
1.7.6 it keeps each reaction system in 65 DEG C, the pipe lid of sample library pipe and Hybridization Buffer liquid pipe is opened, rapidly 13
The hybridization buffer of μ L is transferred in the pipe of sample library, is incubated at least 5min.
1.7.7 it keeps each reaction system in 65 DEG C, opens oligonucleotide library mixing property management lid, rapidly manage sample library
In all reaction systems be transferred completely into oligonucleotide library mixing property management, with pipettor blow and beat mix.
1.7.8 ensure that hybridization reaction solution is all gathered in tube bottom, about 29 μ L of hybrid mixed liquid at this time.
1.7.9 PCR pipe is kept to hybridize 24 hours in 65 DEG C (PCR instrument heat lid is set as 105 DEG C).
It is eluted after 1.8 hybridization: hybrid product being captured using M-280 Streptavidin MagneSphere, then passes through elution
Remove non-capture target gene and other impurity.Specifically comprise the following steps:
1.8.1 constant temperature mixer is adjusted to 65 DEG C in advance, each hybridization reaction, which prepares 1.8mL Wash Buffer#2, to carry out
Preheating；
1.8.2 strepavidin magnetic beads are taken out from refrigerator and the mixing that is vortexed in advance, equilibrium at room temperature 30min；
1.8.3 each hybridization reaction take 50 μ L Dynabeads M-280 Streptavidin MagneSpheres in a new 1.5ml from
Heart pipe；
1.8.4 it takes 200 μ L combination buffers (Binding buffer) to be added in magnetic bead, is acutely vibrated with whirlpool mixed instrument
5 seconds resuspension magnetic beads；
1.8.5 centrifuge tube is placed in 2min on magnetic frame, is clarified completely to liquid；Carefully draws and discard supernatant；
1.8.6 it repeats step 1.8.4 to 1.8.5 twice, washes in total three times.
1.8.7 200 μ L combination buffers (Binding buffer) are added, magnetic bead is resuspended；
1.8.8 after 24 hours are incubated for, hybridization solution, measurement hybridization volume, if steaming the washing and elution of capture dna: are taken out
Hair volume is more than that 8 μ L then think that hybrid experiment fails；
1.8.9 whole hybrid mixed liquid are transferred in ready magnetic bead, piping and druming mixes repeatedly, is closed using sealed membrane
Nozzle；
1.8.10 the centrifuge tube equipped with hybrid mixed liquid and magnetic bead is rotated on vertical blending instrument mixing, mixed at room temperature
At least 45min；
1.8.11 sample is removed from evenly mixing device, removes sealed membrane, it is residual that brief centrifugation 5s ensures that pipe covers no liquid
It stays；
1.8.12 the transfer of 1.5ml centrifuge tube is placed on magnetic frame, stands 3-5min and waits for that liquid is clarified completely, it is careful to inhale
It takes and discards supernatant；
1.8.13 magnetic bead is resuspended with 500 μ L washing buffer (Wash Buffer) #1, and in vibrating 5 on whirlpool mixed instrument
Second mixes sample, at room temperature samples of incubation 15min；
1.8.14 it by centrifuge tube brief centrifugation 3s, is then placed on magnetic frame, stands 3-5min and wait for that liquid is clarified completely,
Carefully draws and discard supernatant；
1.8.15 magnetic bead is resuspended in washing buffer (Wash Buffer) #2 preheated with 65 DEG C of 500 μ L, and mixed in whirlpool
It closes and vibrates 5 seconds on instrument to mix sample, place it in 65 DEG C of incubation 10min in constant temperature mixer；
1.8.16 centrifuge tube is overturned to mix sample, and then brief centrifugation 3s is placed it on magnetic frame, stand 3-
5min waits for that liquid is clarified completely, carefully draws and discards supernatant；
1.8.17 repeat step 1.8.15 to step 1.8.16 twice, altogether wash three times；
1.8.18 magnetic bead is resuspended with the water of 38 μ L nuclease frees.
1.9Post-PCR:
1.9.1 using the DNA hybridization product with M-280 Streptavidin MagneSphere as template, the reactant of configuration such as the following table 10
System carries out PCR amplification, 10 circulations of amplification under corresponding conditions.
Table 10
1.9.2 it is placed in PCR instrument and is reacted according to following procedure: 98 DEG C of 45s；(98 DEG C of 15s, 65 DEG C of 30s, 72 DEG C of 30s) 10
A circulation；72℃1min；4℃∞.
1.9.3PCR after, using Qubit HS Kit quantitative PCR product, if PCR product concentration is lower than 1.6ng/ μ L,
A PCR cycle then is increased to the library lower than this concentration, program is as follows: 98 DEG C of 45s；(98 DEG C of 15s, 65 DEG C of 30s, 72 DEG C
30s) 1 circulation；72℃1min；4℃∞.
The purifying of 1.10Post-PCR product:
1.10.1 30min is balanced at room temperature using preceding be placed in magnetic bead；
1.10.2 the DNA after 100 μ L Post-PCR reaction is transferred in the EP pipe of 1.5mL, the magnetic of 1.5 times of volumes is added
Pearl (150 μ L) blows and beats 10 mixings with pipettor；
1.10.3 standing 10min at room temperature combines magnetic bead sufficiently with DNA, then brief centrifugation 3 seconds；
1.10.4 EP pipe magnetic frame is put into clarify up to liquid；
1.10.5 with the careful removal supernatant of pipettor (being careful not to be drawn onto magnetic bead)；
1.10.6 keep EP pipe on magnetic frame, the ethyl alcohol of 400 μ L 80% of addition, two weeks washing magnetic bead surfaces of tube, with
Remove salt ion and unadsorbed DNA etc.；
1.10.7 it stands, removes ethyl alcohol after solution clarification；
1.10.8 step 1.10.6 to 1.10.7 is repeated；
1.10.9 upper magnetic frame 1min, completely removes ethyl alcohol, and magnetic bead is uncapped and is placed in magnetic frame as far as possible after being slightly centrifuged
On, until magnetic bead surfaces are lackluster (about 10 minutes)；
1.10.10 the water of 52 μ L nuclease frees is added, gently rinse magnetic bead from tube wall and blows and beats 10 mixings；
1.10.11 10min is stored at room temperature so that DNA is eluted from magnetic bead completely；
1.10.12 EP pipe is put into magnetic frame to clarify up to liquid, the 50 μ L DNA eluted is transferred to a new EP
Guan Zhong；
1.10.13Post-PCR product is secondarily purified: balancing 30min at room temperature using preceding be placed in magnetic bead；
1.10.14 by after the EP pipe centrifugation of the DNA equipped with 50 μ L PCR for the first time after purification, the magnetic of 1.5 times of volumes is added
Pearl blows and beats 10 mixings with pipettor；
1.10.15 standing 10min at room temperature combines magnetic bead sufficiently with DNA, then brief centrifugation 3 seconds；
1.10.16 EP pipe magnetic frame is put into clarify up to liquid；
1.10.17 with the careful removal supernatant of pipettor (being careful not to be drawn onto magnetic bead)；
1.10.18 keep EP pipe on magnetic frame, the ethyl alcohol of 400 μ L 80% of addition, two weeks washing magnetic bead surfaces of tube,
To remove salt ion and unadsorbed DNA etc.；
1.10.19 it stands, removes ethyl alcohol after solution clarification；
1.10.20 repeating step 1.10.18 to 1.10.19；
1.10.21 upper magnetic frame 1min, completely removes ethyl alcohol, and magnetic bead is placed in uncap and is placed in magnetic as far as possible after being slightly centrifuged
On power frame, until magnetic bead surfaces are lackluster (about 10 minutes)；
1.10.22 the water of 33 μ L nuclease frees is added, gently rinse magnetic bead from tube wall and blows and beats 10 mixings；
1.10.23 10min is stored at room temperature so that DNA is eluted from magnetic bead completely；
1.10.24 EP pipe is put into magnetic frame to clarify up to liquid, the 31 μ L DNA eluted is transferred to a new EP
Guan Zhong.
1.11 library Quality Controls: using Qubit Instrument measuring library concentration, then dense with Aglient2100 Instrument measuring library
It is whether consistent with the concentration value in the library measured Aglient2100 to compare Qubit instrument for degree and clip size.Specifically include as
Lower step:
1.11.1Qubit HS is quantitative: 1 library μ L being taken to useDsDNA HS Assay Kit is quantitative, records library
Concentration；
1.11.2Agilent 2100 is quantitative: 1 library μ L being taken to use Agilent 2100high sensitivity DNA
Kit detects library size and concentration；
1.11.3 after library detection, it is desirable that between 200bp-400bp, master tape concentrates on the main peak range of library DNA
200-300bp is in good unimodal state, and the primer less than 100bp does not pollute, as a result as shown in Figure 2.
2. machine is sequenced on
The amplification of 2.1 emulsion-based PCRs:
According to library quantitative result, is calculated by each operation (run) loading 5-10pM, library is blended in one by equal proportion
It rises (each run can survey≤16 samples)；Then emulsion-based PCR is carried out by template of mixed library.
Machine is sequenced on 2.2: after emulsion-based PCR, the purifying of PCR reaction solution is carried out according to BGISEQ-100 operational manual
It is sequenced with upper machine.
3. sequencing quality control data
From sequencing quality control data (table 11), the capture rate in library can reach 30% or more, and average sequencing depth can
Up to 400X or more, 98% or more coverage rate, repetitive rate is substantially below 65%.Since FFPE sample quality is irregular, can lead
Quality Control data are caused to have certain fluctuation, but these sequencing quality control data are able to satisfy the requirement of information analysis.
Table 11
4. mutated gene Analysis of test results
From testing result (table 12), the testing result one of low initial amount banking process of the invention and conventional banking process
Cause property is 100%, and the present invention still can be good at detecting simultaneously when Jiang Jianku initial amount is reduced to 100ng or less
Tetra- kinds of mutated genes of SNV, INdel, Fusion, CNV, and to the minimum detection limit of SNV, Indel can reach 3% hereinafter,
Illustrate that this method can be good at the detection for being applied to clinical tumor tissue small sample (as punctured sample).
Table 12
Embodiment two
The positive criteria product that the preparation frequency of mutation is 3% is simulated using tumor cell line DNA and YH DNA, with the standard items
The performance in library of establishing is acted for 50ng sample for the Samples Estimates system.Banking process is the same as embodiment one.Test result is as follows table
13, shown in table 14, from sequencing quality control data (table 13), the capture rate in library can reach 25% or more, and average sequencing is deep
It spends up to 300X or more, 98% or more coverage rate, repetitive rate is substantially below 50%.From the point of view of testing result (table 14), 50ng
It acts library of establishing and is able to detect mutation of the frequency of mutation 3% or more in tumor tissues sample.
Table 13
Table 14
Use above specific case is illustrated the present invention, is merely used to help understand the present invention, not to limit
The system present invention.For those skilled in the art, according to the thought of the present invention, can also make several simple
It deduces, deform or replaces.
SEQUENCE LISTING
<110>Shenzhen Hua Da gene limited liability company, Guangzhou Co., Ltd of Hua Da gene medical test institute
<120>a kind of low initial amount banking process suitable for next-generation microarray dataset
<130> 17I24114
<160> 52
<170> PatentIn version 3.3
<210> 1
<211> 43
<212> DNA
<213>artificial sequence
<400> 1
ccatctcatc cctgcgtgtc tccgactcag ctaaggtaac gat 43
<210> 2
<211> 30
<212> DNA
<213>artificial sequence
<400> 2
atcgttacct tagctgagtc ggagacacgc 30
<210> 3
<211> 43
<212> DNA
<213>artificial sequence
<400> 3
ccatctcatc cctgcgtgtc tccgactcag taaggagaac gat 43
<210> 4
<211> 30
<212> DNA
<213>artificial sequence
<400> 4
atcgttctcc ttactgagtc ggagacacgc 30
<210> 5
<211> 43
<212> DNA
<213>artificial sequence
<400> 5
ccatctcatc cctgcgtgtc tccgactcag aagaggattc gat 43
<210> 6
<211> 30
<212> DNA
<213>artificial sequence
<400> 6
atcgaatcct cttctgagtc ggagacacgc 30
<210> 7
<211> 43
<212> DNA
<213>artificial sequence
<400> 7
ccatctcatc cctgcgtgtc tccgactcag taccaagatc gat 43
<210> 8
<211> 30
<212> DNA
<213>artificial sequence
<400> 8
atcgatcttg gtactgagtc ggagacacgc 30
<210> 9
<211> 43
<212> DNA
<213>artificial sequence
<400> 9
ccatctcatc cctgcgtgtc tccgactcag cagaaggaac gat 43
<210> 10
<211> 30
<212> DNA
<213>artificial sequence
<400> 10
atcgttcctt ctgctgagtc ggagacacgc 30
<210> 11
<211> 43
<212> DNA
<213>artificial sequence
<400> 11
ccatctcatc cctgcgtgtc tccgactcag ctgcaagttc gat 43
<210> 12
<211> 30
<212> DNA
<213>artificial sequence
<400> 12
atcgaacttg cagctgagtc ggagacacgc 30
<210> 13
<211> 43
<212> DNA
<213>artificial sequence
<400> 13
ccatctcatc cctgcgtgtc tccgactcag ttcgtgattc gat 43
<210> 14
<211> 30
<212> DNA
<213>artificial sequence
<400> 14
atcgaatcac gaactgagtc ggagacacgc 30
<210> 15
<211> 43
<212> DNA
<213>artificial sequence
<400> 15
ccatctcatc cctgcgtgtc tccgactcag ttccgataac gat 43
<210> 16
<211> 30
<212> DNA
<213>artificial sequence
<400> 16
atcgttatcg gaactgagtc ggagacacgc 30
<210> 17
<211> 43
<212> DNA
<213>artificial sequence
<400> 17
ccatctcatc cctgcgtgtc tccgactcag tgagcggaac gat 43
<210> 18
<211> 30
<212> DNA
<213>artificial sequence
<400> 18
atcgttccgc tcactgagtc ggagacacgc 30
<210> 19
<211> 43
<212> DNA
<213>artificial sequence
<400> 19
ccatctcatc cctgcgtgtc tccgactcag ctgaccgaac gat 43
<210> 20
<211> 30
<212> DNA
<213>artificial sequence
<400> 20
atcgttcggt cagctgagtc ggagacacgc 30
<210> 21
<211> 43
<212> DNA
<213>artificial sequence
<400> 21
ccatctcatc cctgcgtgtc tccgactcag tcctcgaatc gat 43
<210> 22
<211> 30
<212> DNA
<213>artificial sequence
<400> 22
atcgattcga ggactgagtc ggagacacgc 30
<210> 23
<211> 43
<212> DNA
<213>artificial sequence
<400> 23
ccatctcatc cctgcgtgtc tccgactcag taggtggttc gat 43
<210> 24
<211> 30
<212> DNA
<213>artificial sequence
<400> 24
atcgaaccac ctactgagtc ggagacacgc 30
<210> 25
<211> 43
<212> DNA
<213>artificial sequence
<400> 25
ccatctcatc cctgcgtgtc tccgactcag tctaacggac gat 43
<210> 26
<211> 30
<212> DNA
<213>artificial sequence
<400> 26
atcgtccgtt agactgagtc ggagacacgc 30
<210> 27
<211> 43
<212> DNA
<213>artificial sequence
<400> 27
ccatctcatc cctgcgtgtc tccgactcag ttggagtgtc gat 43
<210> 28
<211> 30
<212> DNA
<213>artificial sequence
<400> 28
atcgacactc caactgagtc ggagacacgc 30
<210> 29
<211> 43
<212> DNA
<213>artificial sequence
<400> 29
ccatctcatc cctgcgtgtc tccgactcag tctagaggtc gat 43
<210> 30
<211> 30
<212> DNA
<213>artificial sequence
<400> 30
atcgacctct agactgagtc ggagacacgc 30
<210> 31
<211> 43
<212> DNA
<213>artificial sequence
<400> 31
ccatctcatc cctgcgtgtc tccgactcag tctggatgac gat 43
<210> 32
<211> 30
<212> DNA
<213>artificial sequence
<400> 32
atcgtcatcc agactgagtc ggagacacgc 30
<210> 33
<211> 43
<212> DNA
<213>artificial sequence
<400> 33
ccatctcatc cctgcgtgtc tccgactcag tctattcgtc gat 43
<210> 34
<211> 30
<212> DNA
<213>artificial sequence
<400> 34
atcgacgaat agactgagtc ggagacacgc 30
<210> 35
<211> 43
<212> DNA
<213>artificial sequence
<400> 35
ccatctcatc cctgcgtgtc tccgactcag aggcaattgc gat 43
<210> 36
<211> 30
<212> DNA
<213>artificial sequence
<400> 36
atcgcaattg cctctgagtc ggagacacgc 30
<210> 37
<211> 43
<212> DNA
<213>artificial sequence
<400> 37
ccatctcatc cctgcgtgtc tccgactcag ttagtcggac gat 43
<210> 38
<211> 30
<212> DNA
<213>artificial sequence
<400> 38
atcgtccgac taactgagtc ggagacacgc 30
<210> 39
<211> 43
<212> DNA
<213>artificial sequence
<400> 39
ccatctcatc cctgcgtgtc tccgactcag cagatccatc gat 43
<210> 40
<211> 30
<212> DNA
<213>artificial sequence
<400> 40
atcgatggat ctgctgagtc ggagacacgc 30
<210> 41
<211> 43
<212> DNA
<213>artificial sequence
<400> 41
ccatctcatc cctgcgtgtc tccgactcag tcgcaattac gat 43
<210> 42
<211> 30
<212> DNA
<213>artificial sequence
<400> 42
atcgtaattg cgactgagtc ggagacacgc 30
<210> 43
<211> 43
<212> DNA
<213>artificial sequence
<400> 43
ccatctcatc cctgcgtgtc tccgactcag ttcgagacgc gat 43
<210> 44
<211> 30
<212> DNA
<213>artificial sequence
<400> 44
atcgcgtctc gaactgagtc ggagacacgc 30
<210> 45
<211> 43
<212> DNA
<213>artificial sequence
<400> 45
ccatctcatc cctgcgtgtc tccgactcag tgccacgaac gat 43
<210> 46
<211> 30
<212> DNA
<213>artificial sequence
<400> 46
atcgttcgtg gcactgagtc ggagacacgc 30
<210> 47
<211> 43
<212> DNA
<213>artificial sequence
<400> 47
ccatctcatc cctgcgtgtc tccgactcag aacctcattc gat 43
<210> 48
<211> 30
<212> DNA
<213>artificial sequence
<400> 48
atcgaatgag gttctgagtc ggagacacgc 30
<210> 49
<211> 41
<212> DNA
<213>artificial sequence
<400> 49
ccactacgcc tccgctttcc tctctatggg cagtcggtga t 41
<210> 50
<211> 43
<212> DNA
<213>artificial sequence
<400> 50
atcaccgact gcccatagag aggaaagcgg aggcgtagtg gtt 43
<210> 51
<211> 20
<212> DNA
<213>artificial sequence
<400> 51
ccatctcatc cctgcgtgtc 20
<210> 52
<211> 28
<212> DNA
<213>artificial sequence
<400> 52
ccactacgcc tccgctttcc tctctatg 28
Claims (10)
1. a kind of low initial amount banking process suitable for next-generation microarray dataset, which comprises the steps of:
(1) DNA of extraction DNA fragmentation: is broken into segment；
(2) end is repaired: the DNA fragmentation interrupted being added to end and is repaired in reaction solution, is incubated for；
(3) it purifies for the first time: repairing product with magnetic beads for purifying end, and retain magnetic bead in system；
(4) connector connects: product being repaired in end after purification and is added in connection reaction solution together with magnetic bead, is added simultaneously
Connector is incubated for；
(5) it purifies for second: twice with magnetic beads for purifying connector connection product, and removing magnetic bead；
(6) first time PCR amplification: using the connector connection product of purifying as template, PCR amplification is carried out；
(7) segment is screened: screening first time pcr amplification product with magnetic bead；
(8) hybridize: the first time pcr amplification product that segment is screened is hybridized with chip；
(9) it captures and elutes: hybrid product being captured with magnetic bead, then gone by elution unless capturing target gene and its
Its impurity；
(10) second of PCR amplification: PCR amplification is carried out as template using the DNA with magnetic bead after eluting, purpose is further amplified
Gene signal；
(11) third time purifies: twice with second of pcr amplification product of magnetic beads for purifying.
2. the method according to claim 1, wherein DNA derives from tumor puncture sample in the step (1).
3. the method according to claim 1, wherein the amount of DNA is 10ng to 100ng in the step (1).
4. the method according to claim 1, wherein it is 200bp left that DNA, which is broken into size, in the step (1)
Right segment.
5. the method according to claim 1, wherein pure with 1.8 times of AMPure XP magnetic beads in the step (3)
Change end and repairs product.
6. the method according to claim 1, wherein pure with 1.2 times of AMPure XP magnetic beads in the step (5)
Change connector connection product.
7. the method according to claim 1, wherein respectively with 0.8 times and 0.2 times in the step (7)
AMPure XP magnetic bead screens first time pcr amplification product.
8. the method according to claim 1, wherein first time pcr amplification product and chip in the step (8)
Before being hybridized, is calculated by the DNA total amount of every chip hybridization 800ng, 4-8 sample is mixed into a text in proportion
Library.
9. the method according to claim 1, wherein with Streptavidin MagneSphere to hybridization in the step (9)
Product is captured.
10. the method according to claim 1, wherein with 1.5 times of AMPure XP magnetic beads in the step (11)
Purify second of pcr amplification product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710551477.2A CN109207557A (en) | 2017-07-07 | 2017-07-07 | A kind of low initial amount banking process suitable for next-generation microarray dataset |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710551477.2A CN109207557A (en) | 2017-07-07 | 2017-07-07 | A kind of low initial amount banking process suitable for next-generation microarray dataset |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109207557A true CN109207557A (en) | 2019-01-15 |
Family
ID=64991098
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710551477.2A Pending CN109207557A (en) | 2017-07-07 | 2017-07-07 | A kind of low initial amount banking process suitable for next-generation microarray dataset |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109207557A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111041069A (en) * | 2019-12-26 | 2020-04-21 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
| CN112430666A (en) * | 2020-12-22 | 2021-03-02 | 武汉艾迪康医学检验所有限公司 | Method for detecting tumor-associated polygene mutation by high-throughput sequencing |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102485979B (en) * | 2010-12-02 | 2014-02-19 | 深圳华大基因科技服务有限公司 | Formalin-fixed paraffin-embedded (FFPE) sample nucleic acid library, construction method and FFPE sample analysis method |
| CN104946629A (en) * | 2015-07-14 | 2015-09-30 | 天津诺禾医学检验所有限公司 | Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample |
| CN104894651B (en) * | 2015-06-29 | 2017-04-12 | 天津诺禾医学检验所有限公司 | Building method of high-throughput sequencing library of trace starter DNA (deoxyribonucleic acid) and high-throughput sequencing library built by building method |
-
2017
- 2017-07-07 CN CN201710551477.2A patent/CN109207557A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102485979B (en) * | 2010-12-02 | 2014-02-19 | 深圳华大基因科技服务有限公司 | Formalin-fixed paraffin-embedded (FFPE) sample nucleic acid library, construction method and FFPE sample analysis method |
| CN104894651B (en) * | 2015-06-29 | 2017-04-12 | 天津诺禾医学检验所有限公司 | Building method of high-throughput sequencing library of trace starter DNA (deoxyribonucleic acid) and high-throughput sequencing library built by building method |
| CN104946629A (en) * | 2015-07-14 | 2015-09-30 | 天津诺禾医学检验所有限公司 | Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample |
Non-Patent Citations (2)
| Title |
|---|
| SONJA BERENSMEIER: "Magnetic particles for the separation and purification of nucleic acids", 《APPL MICROBIOL BIOTECHNOL》 * |
| 韩云霞: "DNA常见纯化方法比较及分析综述", 《生物技术世界》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111041069A (en) * | 2019-12-26 | 2020-04-21 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
| CN111041069B (en) * | 2019-12-26 | 2021-01-19 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
| CN112430666A (en) * | 2020-12-22 | 2021-03-02 | 武汉艾迪康医学检验所有限公司 | Method for detecting tumor-associated polygene mutation by high-throughput sequencing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107002292B (en) | A kind of construction method and reagent in the twin adapter single stranded circle library of nucleic acid | |
| CN106987905A (en) | A kind of construction method and kit in BRCA1/2 genetic tests library | |
| WO2012068919A1 (en) | Dna library and preparation method thereof, and method and device for detecting snps | |
| CN107858414A (en) | A kind of high-flux sequence joint, its preparation method and its application in ultralow frequency abrupt climatic change | |
| CN112195521A (en) | DNA/RNA co-database building method based on transposase, kit and application | |
| CN111979307B (en) | Targeted sequencing method for detecting gene fusion | |
| CN106835291B (en) | The preparation method and kit of DNA library | |
| CN109576347A (en) | The sequence measuring joints of the label containing unimolecule and the construction method of sequencing library | |
| CN110004225B (en) | Tumor chemotherapeutic drug individualized gene detection kit, primers and method | |
| CN112662760A (en) | Cancer gene methylation detection system and cancer in-vitro detection method implemented in cancer gene methylation detection system | |
| CN111041069B (en) | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof | |
| CN107619867A (en) | For detecting the combined sequence and probe of lung cancer several genes mutation type simultaneously | |
| CN109234357A (en) | It is a kind of for detecting whether target gene occurs the method for fusion mutation, primer combination, kit and its application | |
| CN112662762A (en) | Probe composition for detecting 5 tumors of digestive tract | |
| CN111926066A (en) | Methods and kits for rapid hybridization of probes to libraries | |
| CN110791500B (en) | Probe composition and kit for detecting lung cancer mutant gene based on NGS method | |
| CN109207557A (en) | A kind of low initial amount banking process suitable for next-generation microarray dataset | |
| CN107603971A (en) | A kind of preparation method of in situ hybridization probe | |
| CN107988320A (en) | A kind of molecular label connector and its preparation method and application | |
| TW201321520A (en) | Method and system for virus detection | |
| CN112259165A (en) | Method and system for detecting microsatellite instability state | |
| CN110241211B (en) | Molecular combination marker for diagnosing colorectal cancer and detection kit | |
| CN114058706B (en) | Detection primer for detecting lung cancer gene by using free DNA and kit thereof | |
| CN110273028A (en) | Enrichment method, sequencing data analysis method and the device of viral integrase type DNA | |
| CN116536308A (en) | Sequencing sealant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190115 |