CN106867996A - A kind of cfDNA libraries of the method, kit and structure that cfDNA is extracted from hydrothorax - Google Patents
A kind of cfDNA libraries of the method, kit and structure that cfDNA is extracted from hydrothorax Download PDFInfo
- Publication number
- CN106867996A CN106867996A CN201710117869.8A CN201710117869A CN106867996A CN 106867996 A CN106867996 A CN 106867996A CN 201710117869 A CN201710117869 A CN 201710117869A CN 106867996 A CN106867996 A CN 106867996A
- Authority
- CN
- China
- Prior art keywords
- cfdna
- hydrothorax
- sample
- wash solution
- dna
- 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.)
- Granted
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
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
-
- 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
- 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
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
-
- 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
Abstract
CfDNA is extracted from hydrothorax sample the present invention relates to one kind, the method for building high-throughput sequencing library belongs to technical field of gene detection.The present invention has successfully extracted cfDNA, the samples sources that empirical tests can build as high-throughput sequencing library from hydrothorax first, and can turn into one kind effectively supplement of blood plasma cfDNA；In addition, after cfDNA sizes fragment is separated in hydrothorax sample in the method, so that the cfDNA purity in hydrothorax sample is higher, make it more suitable for the structure of DNA library, the more conducively detection of later stage mutation, can solve the problems, such as the conventional sample unicity and recall rate that storehouse is only built by blood plasma extraction cfDNA.
Description
Technical field
The present invention relates to a kind of cfDNA libraries of the method, kit and structure that cfDNA is extracted from hydrothorax, belong to
Technical field of gene detection.
Background technology
With being continuously increased for morbidity and mortality, cancer has become the dead first cause of Chinese population and main
Public health problem.Counted according to National Cancer Center, China there are about 4,290,000 cancer new cases, 2,810,000 within 2015
Cancer death, lung cancer turns into most common cancer, is also the first cause of cancer mortality.Along with various treatment means
Constantly rise, such as radiotherapy of early stage, the chemotherapy in modern age targeted therapy till now and immunization therapy, the treatment of cancer just gradually to
Precision Therapeutic mode develops, and is especially targeted treatment and immunization therapy, and the thus screening for selectively targeted crowd seems
It is particularly important, and used as clinical supplementary means indispensable in targeted drug therapy, the genetic test of cancer is directly connected to
Therapeutic efficiency of the medicine for patient.
High throughput sequencing technologies are also called sequencing technologies of future generation, due to its high flux, the characteristic such as sensitivity is high, in cancer
It is used widely in genetic test.Used as the mostly important ring of high-flux sequence, the preparation of library sample seems particularly heavy
Will, current commonplace detection sample has the tumor tissues sample of patient, punctures sample etc., but these are all undoubtedly
Belong to invasive.And with advances in technology, plasma sample is also gradually applied in high-flux sequence, plasma DNA
（Cell free DNA, cf-DNA）, it is the free DNA for existing in blood plasma, cfDNA is damaged from body cell, and what is had comes from
In normal cell, what is had comes from abnormal cell（Such as tumour cell）.For Healthy People, the cfDNA main sources in its blood
It is normally metabolic body cell, therefore a relatively low level should be maintained.However, work as having abnormal cell（Tumour is thin
Born of the same parents）When forming or have very serious systemic organ damage, the cell of mortality（Either apoptosis or necrosis）Can produce
The substantial amounts of cfDNA of life.And its non-invasive characteristic is praised highly extensively, the biopsy of high-flux sequence combination blood plasma ctDNA liquid can be with
Realize the dynamic detection to tumor patient.But the detection of plasma sample mutation, is influenceed, the shape residing for tumour by many factors
The current stable disease of state such as patient still enters exhibition influences the detection of the peculiar mutation of tumour, and such cancer staging, cancer turns
Position of shifting etc. may all have influence on the detection of ctDNA in blood plasma, therefore a kind of supplement of liquid biopsy sample of exigence.
Most commonly seen in the main late lung cancer of hydrothorax, other tumours such as breast cancer, lymthoma etc. may all be transferred to chest
Abdominal cavity and cause cancerous hydrothorax, the incidence of Malignant Pleural is up to 60% in lung cancer, and pathogenic factor is mainly pleura metastasis tubercle
Invade and block caused by capillary and lymphatic vessel.
The content of the invention
Explored by lot of experiments, the present invention have unexpectedly discovered that a kind of can extraction from hydrothorax obtains higher degree
The method and dedicated kit of cfDNA, can be optimized using the technology and extract cfDNA recall rates not from blood plasma in the prior art
The problem of foot；And after processing sample, the structure of DNA library can be effectively realized, this method is extracted
The cfDNA for obtaining by high-flux sequence, can effectively detect the mutation of patient's carrying, and it was found that in this way
The cfDNA that obtains is built after the sequencing of library, during recall rate of its mutation is higher than blood/plasma sample in some samples
CfDNA, so that more can effectively point out patient prognosis, the information such as medication.
The first aspect of the invention：
There is provided a kind of method that cfDNA is extracted from hydrothorax, comprise the following steps：
1st step, is centrifuged to hydrothorax sample；
2nd step, adds Carrier RNA, Proteinase K, lysate in the supernatant that the 1st step is obtained, and carries out incubation treatment, then add
Enter DNA and separate out liquid；
3rd step, the sample that the 2nd step is obtained is adsorbed by the way of post absorption to cfDNA, then is passed through after desorption, is obtained
Get cfDNA.
In one embodiment, 50~150 mmol Tris-HCl, 20~30 mmol are included in described lysate
EDTA, 400~600 mmol NaCl, 0.5~1.5% SDS.
In one embodiment, described DNA includes 0.1~0.2 mol/L NaCl, 0.02~0.05 in separating out liquid
M phosphate solutions.
In one embodiment, pellosil adsorption column is used in post absorption.
In one embodiment, before being desorbed, successively using salt wash solution and alcohol wash solution to adsorption column
It is rinsed.
Included in described salt wash solution：10~20 mM trishydroxymethylaminomethanes, 1~5 M guanidine hydrochlorides, 10
~30 mM EDTA-2Na, 0.1~1% HCl.
Described alcohol wash solution is by the ethanol and isopropanol 1 of 75~80vol.%:1 prepares.
The addition of described Carrier RNA is that 5~50 μ l are added in every milliliter of hydrothorax sample, Proteinase K plus
It is that 50~500 μ l are added in every milliliter of hydrothorax sample to enter amount, the addition of lysate be add 0.5 in every milliliter of hydrothorax sample~
The addition that 5ml, DNA separate out liquid is 0.5~5ml of addition in every milliliter of hydrothorax sample；The consumption of salt wash solution is every milliliter
30~100 μ l of hydrothorax sample correspondence；The consumption of alcohol wash solution is every milliliter of 150~300 μ l of hydrothorax sample correspondence.
The used stripping liquid of desorption is without enzyme water.
The second aspect of the invention：
There is provided a kind of kit that cfDNA is extracted from hydrothorax, include：Lysate, DNA separate out liquid, Proteinase K,
Carrier RNA, adsorption column, salt wash solution, alcohol wash solution, stripping liquid.
Lysate, DNA separate out liquid, Proteinase K, Carrier RNA, salt wash solution, the volume ratio model of alcohol wash solution
Enclosing is：500~5000：500~5000：50~500：5~50：30~100：150~300.
Described stripping liquid is without enzyme water.
The third aspect of the invention：
CfDNA libraries are obtained there is provided by extracting the cfDNA for obtaining structures from hydrothorax.
The fourth aspect of the invention：
There is provided application of the above-mentioned cfDNA libraries in gene sequencing.
In one embodiment, described gene sequencing is to use NGS（Next generation's sequencing）Method.
The fifth aspect of the invention：
There is provided a kind of construction method in cfDNA libraries, comprise the following steps：
1st step, size fragment separation is carried out to the cfDNA that extraction is obtained；
2nd step, end reparation, 3' ends is carried out to small pieces cfDNA and, plus joint, purifying, is obtained plus base A, two ends successively
To cfDNA libraries.
In one embodiment, the size fragment in the 1st described step is separated is separated using paramagnetic particle method.
The sixth aspect of the invention：
The present invention also provides a kind of cfDNA library construction Kits and its application in gene sequencing, is included in the kit
The kit of said extracted cfDNA.
Beneficial effect
The present invention has successfully extracted cfDNA, the sample that empirical tests can build as high-throughput sequencing library from hydrothorax first
This source, and one kind effectively supplement of blood plasma cfDNA can be turned into；Additionally, by hydrothorax sample in the method
After cfDNA sizes fragment is separated so that the cfNDA purity in hydrothorax sample is higher, makes it more suitable for the structure of DNA library,
The more conducively detection of later stage mutation, can solve the conventional sample unicity and recall rate that storehouse is only built by blood plasma extraction cfDNA
Problem.
Brief description of the drawings
Fig. 1 is hydrothorax supernatant cfDNA by the unsegregated Quality Control figure of size fragment；
Fig. 2 be hydrothorax supernatant cfDNA by size fragment by the Quality Control figure after separation；
Fig. 3 is the Quality Control figure that the cfDNA obtained by Beads enrichment in embodiment 2 builds library；
Specific embodiment
The present invention is described in further detail below by specific embodiment.But those skilled in the art will manage
Solution, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted specific skill in embodiment
Art or condition person, are carried out according to the technology or condition described by document in the art or according to product description.Examination used
Agent or the unreceipted production firm person of instrument, be can by city available from conventional products.Word used herein " including ",
"comprising", " having " or its any other variant are intended to cover including for non-exclusionism.For example, including list key element technique,
Method, article or equipment are not necessarily limited by those key elements, and can be to be not expressly set out including other or belong to this work
Skill, method, article or the intrinsic key element of equipment.Unless context clear stipulaties, otherwise singulative "/kind " and " described
(being somebody's turn to do) " includes a plurality of discussion objects, and heretofore described percentage refers to quality percentage in the case of without special instruction
Than.
" hydrothorax sample " described in following examples refers to original acquirement hydrothorax sample in the case of without special instruction
This, with the consumption of reagent is with initial hydrothorax sample volume as standard stabiliser in extraction process.
Have passed through lot of experiments to grope, the present invention have unexpectedly discovered that a kind of method for obtaining cfDNA from hydrothorax, this
Method can preferably obtain the cfDNA for being suitable for library construction from hydrothorax, and this extracting method is mainly by centrifugation, egg
White enzyme K treatment, the method for post absorption obtain the cfDNA in hydrothorax.
The extraction of 1.cfDNA
1.1 centrifugations：
In centrifugation step, main purpose is to separate cell, high molecular weight protein, blood formed element in hydrothorax etc., is obtained
Supernatant, present invention finds cfDNA is contained in supernatant, these cfDNA can compare blood by being suitable for library construction after extraction
The cfDNA that liquid sample is obtained has mutation recall rate higher.Here centrifugation step, can in 1000~20000 rpm from
The min of the heart 5~30, supernatant is in 4 DEG C of preservations.
The treatment of 1.2 protein breakdowns：
Addition Carrier RNA are also needed in hydrothorax supernatant can help nucleic acid to remove electrostatic effect, can very well be adsorbed to purifying
On post, elution efficiency can be improved, so as to reach recovery sample amplifying nucleic acid as big as possible, next needed by proteases for decomposing
Treatment, using Proteinase K to sample incubation treatment, Proteinase K can decompose the protein in supernatant, be incubated the temperature of process
Can control in 45 DEG C of water-bath 10min；Needed after treatment terminates, in sample add lysate, the lysate for using here into
Point can be comprising 50~150 mmol Tris-HCl, 20~30 mmol EDTA, 400~600 mmol NaCl, 0.5~
1.5% SDS；After incubation terminates, in addition it is also necessary to add DNA and separate out liquid to maintain sample system, a low-salt environment can be provided,
Be conducive to the precipitation of dissociative DNA, additionally, promoting the salt of residual protein molten by less salt, and residual protein removed, after reduction
The pollution of continuous protein, described DNA includes 0.1~0.2 mol/L NaCl, 0.02~0.05 M phosphate in separating out liquid
Solution.
1.3 posts are adsorbed：
After the sample after obtaining Proteinase K treatment, post adsorption operations are ready for, present invention discover that passing through this post absorption side
Method can obtain more high-quality cfDNA relative to other methods, and adsorption column adopted here can use pellosil
Post, after adsorption process terminates, is rinsed using cleaning solution, can effectively be got rid of the impurity in sample and be retained more
CfDNA, in a preferred embodiment, can successively be rinsed using two kinds of lavation buffer solutions, and the composition of salt wash solution is：10
~20 mM trishydroxymethylaminomethanes, 1~5 M guanidine hydrochlorides, 10~30 mM EDTA-2Na, 0.1~1% HCl, alcohol washing
The composition of solution is：The ethanol and isopropanol 1 of 75~80vol.%:1 compounding.
After impurity is washed, the cfDNA for adsorbing is eluted using stripping liquid, obtain the wash-out containing cfDNA
Liquid, eluent here is preferably used without enzyme water.
2. library construction：
After obtaining cfDNA, after being built library, the mutation recall rate higher than plasma sample can be shown.Here
The method for building library, can include carries out size fragment separation to the cfDNA that extraction is obtained；To small pieces cfDNA successively
End reparation, 3' ends are carried out plus base A, two ends plus steps such as joint, purifying.
2.1 Beads enrichments
Wherein, the cfDNA that extraction is obtained carry out size fragment to be separated is committed step, by big to cfDNA in hydrothorax sample
After small fragment is separated so that the cfNDA purity in hydrothorax sample is higher, it is to avoid big segment cfDNA makes for the occupancy of flux
Its more suitable for DNA library structure, the more conducively detection of later stage mutation, the abundance of detection can be higher also more credible, can be with
Solve the problems, such as the conventional sample unicity and recall rate that storehouse is only built by blood plasma extraction cfDNA.The separation of size fragment here
Can be adsorbed by cfDNA by the way of Beads enrichment, then after large fragment is washed, by small fragment cfDNA from magnetic bead
Wash-out.
Repair 2.2 ends
CfDNA fragments are carried out into end to repair to enter using Klenow fragments, T4DNA polymerases and T4 polynucleotide kinases
OK, wherein, the Klenow fragments have 5 ' -3 ' ' polymerase activities and 3 ' -5 ' polymerase activities, but lack 5 ' -3 ' excision enzyme
Activity.Thereby, it is possible to easily and accurately carry out end reparation to cfDNA fragments.Embodiments in accordance with the present invention, can also enter one
Step includes the step of being purified to the cfDNA fragments repaired by end, thus, it is possible to easily carry out subsequent treatment.
Using T4 polymerases and Klenow Escherichia coli polymerase segments, for cfDNA 5' protrude viscous end-filling and
3' protrudes viscous end and ties, and produces flat end, is connected for follow-up flush end.Reaction is carried out in PCR amplification instrument, and 20 is Celsius
Degree, 30 minutes.
Table 1
2.3cfDNA sample 3' ends add base A
Base A is added in 3 ' ends of the cfDNA fragments repaired by end, to obtain the cfDNA pieces with cohesive end A
Section.According to one embodiment of present invention, it is possible to use Klenow (3 ' -5 ' exo-), i.e., with 3 ' -5 ' 5 prime excision enzyme activity
Klenow, base A is added in 3 ' ends of the cfDNA fragments repaired by end.Thereby, it is possible to easily and accurately by base A
It is added to 3 ' ends of the cfDNA fragments repaired by end.Embodiments in accordance with the present invention, can further include to tool
The step of DNA fragmentation of toughness end A is purified, thus, it is possible to easily carry out subsequent treatment.
Reaction is carried out in PCR amplification instrument, 37 DEG C, 30 minutes.
Table 2
CDNA/DNA crosses post purifying, commercialization company purification kit.
2.4cfDNA two ends add joint
Table 3
2.5 DNA amplification templates
In one embodiment of the invention, cfDNA fragment amplifications enable that the amount of nucleic acid meets chip/probe hybrid capture
Demand.
Polymerase chain reaction（PCR）, carried out in PCR amplification instrument.
Table 4
PCR conditions：It is placed in PCR amplification instrument, 98 DEG C of predegenerations 30 seconds, 98 DEG C are denatured 30 seconds, and 65 DEG C are annealed 30 seconds, 72 DEG C of extensions
30 seconds, circulate 4-6 times altogether.It is last to extend 5 minutes at 72 DEG C.
The cfDNA libraries that the above method is obtained can apply in the gene sequencing of non-treatment and diagnostic purpose, particularly
Suitable for the sequencing of NGS methods.Above-mentioned cfDNA extracts kits can be applied in library construction Kit, be equally applicable
In gene sequencing.
The acquisition of cfDNA in the hydrothorax sample of embodiment 1
The separation of 1 hydrothorax sample supernatant precipitation
The hydrothorax sample of fresh collection is taken, 1800rpm is centrifuged 10 min, hydrothorax supernatant is adsorbed in new centrifuge tube, discards
Hydrothorax is precipitated, by hydrothorax supernatant Sample preservation to 4 DEG C（-80℃）Refrigerator.
The post adsorbing separation of cfDNA in 2 hydrothorax supernatants
2.1 samplings：Take out 4 DEG C（-80℃）The hydrothorax supernatant sample of preservation, then by 4 DEG C of 16000rpm 10 min treatment；
2.2 configuration premixed liquids：Prepare lysate Buffer ACL Mix（QIAGEN）, draw 2 times of Buffer of hydrothorax cumulative volume
ACL Mix, overturn and mix；
2.3 add Proteinase K：20 μ l Carrier RNA are added in every milliliter of hydrothorax sample, 200 μ l Proteinase Ks are added, filled
Divide digestion；
2.4 add ACL：By adding 2 ml lysate Buffer ACL Mix in every milliliter of hydrothorax sample（QIAGEN）, 45 DEG C of water
Bath 10min, incubation terminates, normal temperature cooling；
2.5 add ACB：Liquid Buffer ACB are separated out by 2ml DNA are added in every milliliter of hydrothorax sample（QIAGEN）, overturn mixed
It is even；
2.6 prepare pump：Rule plug is removed, QIAamp Vac are plugged, connexon is plugged, pillar is loaded onto, closed with covers is kept
State；
2.7 cross post：50ml mixed liquors are transferred in sample tube, vavuum pump are opened, at the uniform velocity by pellosil adsorption column
（QIAGEN）；
2.8 wash-outs：300 μ l salt wash solution Buffer ACW1 are added to adsorption column（QIAGEN）, vavuum pump is opened, slowly
Evenly by adsorption column, 1000 μ l alcohol wash solution Buffer ACW2 are continuously added（QIAGEN）；Continue to be added to adsorption column
450 μ l absolute ethyl alcohols to wash-out terminates, and closes vavuum pump；
2.9 samples are centrifuged：Adsorption column lid is covered, is placed in centrifuge tube, 3000rpm centrifugations 3min；2.10 reclaim
QIAamp vac, plug Rule plug；
2.11 eluted dnas：Add 5-10 μ l without enzyme water to adsorption column, be transferred to for the DNA after wash-out by 1800rpm centrifugation 30s
In new 1.5ml adsorption tubes, 30 μ L being added without enzyme water, standing 3min, 2500rpm centrifugation 5min 2.12 DNA are mixed：With
Vortex is vortexed and mixes.
3 cfDNA samples are quantified
By the DNA sample after mixing, take 1 μ L carry out Qubit quantify.
1 phenol-chloroform of reference examples-isoamyl alcohol extraction method extracts cfDNA
1. take hydrothorax 4ml, 2000rpm room temperature and 10 min are centrifuged, during supernatant moved into new centrifuge tube, then through 12000rpm room temperatures
After centrifugation 5min, during supernatant moved into new centrifuge tube.
2. the μ l of 1%SDS solution 350ml, 20mg/ml Proteinase K Solution 20 will be added in supernatant, and whirlpool concussion is mixed, 58 DEG C
The min of water-bath 75, adds the μ l of phenol-chloroform-isoamyl alcohol mixed liquor 700, and whirlpool concussion is mixed, 12000rpm centrifugations 10min at 4 DEG C,
During supernatant moved into new centrifuge tube, the μ l of isopropanol 800 are added, overturn and mix, 12000rpm centrifugations 10min at 4 DEG C, in tipping
Clearly, 75% ethanol 1ml is added, is overturned and is mixed, 12000rpm centrifugations 10min at 4 DEG C, tipping supernatant, 65 DEG C of metal baths are until remaining
Liquid is completely dried, and adds deionized water 10 μ l, 65 DEG C of dissolving 10min, -20 DEG C of preservations.
2 phenol-chloroforms of reference examples-isoamyl alcohol extraction coordinates minim DNA settling agent method to extract cfDNA
Difference with reference examples 1 is：The μ l of minim DNA settling agent 5 are added while 800 μ l of isopropanol are added.
Reference examples 3
Difference with embodiment 1 is：The addition volume of the first buffer solution is added with hydrothorax supernatant sample equivalent.
Reference examples 4
Difference with embodiment 1 is：DNA is not added to separate out liquid.
Using the DNA sample obtained in above-described embodiment 1 and reference examples 1~4, take 1 μ l carry out Qubit quantify.
The DNA sample quantitative result of table 5
As can be seen from the above table, the cfDNA samples of high content can be obtained from hydrothorax using the method for the present invention, better than normal
The DNA that do not add in rule method and reference examples method, wherein reference examples 4 separates out liquid treatment, can not only improve DNA content
DNA mass is set to be improved, thus it is speculated that its effect includes 1, separates out protein；2nd, remaining protein is removed, makes absorbance ratio
Value A260/A280 meets the requirements between 1.8 to 2.
The structure of the DNA library of embodiment 2
The separation of 3.1 cfDNA size fragments
A) embodiment 1 is extracted into cfDNA samples to add without enzyme water polishing to 50 μ l, adds 35 μ l magnetic bead Axygen beads, use
Liquid-transfering gun is mixed；
B) sample is placed on magnetic separation frame up in supernatant clarification, absorption supernatant to the low adsorption tubes of another 1.5ml；
C) add the resuspended liquid-transfering guns of 65 μ l magnetic bead Axygen beads that the up to supernatant clarification of magnetic separation frame is placed in after mixing, lose
Supernatant is abandoned, the cleaning of the ethanol of 50 μ l 75% is added, liquid-transfering gun is removed raffinate, dried；
D) plus 56 μ L are without enzyme water, mix, in pipetting 51 μ l clarified solutions to new 1.5ml centrifuge tubes；
e）Take 1 μ l Qubit measurement DNA concentrations.The μ l ctDNA small fragments of gained 50 are directly used in cfDNA KAPA and build storehouse stream
Journey.
Magnetic bead size fragment separates front and rear cfDNA Quality Controls figure as depicted in figs. 1 and 2, it can be seen that by magnetic
After pearl separates, size fragment has obtained preferable separation, and the cfDNA contents of small-molecular-weight are significantly improved.
3.2 cfDNA duplex ends are repaired
50 μ L cfDNA after by constant volume all add the reaction system of table 6, and 20 DEG C of reactions in PCR instrument are placed on after mixing
30min, 65 DEG C of reaction 30min.
Table 6cfDNA duplex ends repair reaction system
3.3 Adaptor are linked
A) joint coupled reaction is configured
The joint coupled reaction reaction system of table 7
b）Brief centrifugation is simultaneously fully mixed with p200.20 DEG C, 60min in PCR instrument.
3.4 product purifications
A) the resuspended Axygen beads of 50 μ L, liquid-transfering gun is added to mix, 5min on magnetic separation frame is until supernatant clarification, abandons
Supernatant, plus the ethanol of 50 μ L 75% is washed 2 times, removes residul liquid-removing；
E) add 30 μ L without enzyme water, liquid-transfering gun is mixed, 5min on magnetic frame；
g）Take 1 μ L carry out Qubit quantify, remaining together with beads enter following PCR
3.5 PCR expand enriched product
The supernatant that above-mentioned steps are obtained adds amplification system, and Ju Ti Pei Fang is shown in Table 8, after mixing, reaction system is placed
In PCR instrument, specific response procedures are shown in Table 9.
The amplification enrichment reaction system of table 8
Table 9PCR amplification enrichment reaction conditions
3.6 product purifications
A) add 25 μ L magnetic bead Axygen beads resuspended in above-mentioned product, liquid-transfering gun is mixed, and magnetic frame is up to gone up limpid
Clearly, supernatant is abandoned, adds the ethanol of 50 μ L 75% to clean 2 times, room temperature dries 5min；
b）Plus 31 μ L without enzyme water, liquid-transfering gun is mixed, and is incubated at room temperature 20min, and clarified solution is moved into the low absorption EP of new 1.5ml
Guan Zhong.
c）Take 1 μ L carry out survey Qubit quantify, remaining sample enters follow-up process as sequencing library.
The Quality Control figure in the library for obtaining is as shown in Figure 3.As can be seen that the cfDNA structures obtained using extracting method of the present invention
Building library has more preferable base quality.
Mutation recall rate experiment
In order to probe into whether hydrothorax sample can randomly select 34 patients with lung cancer as the supplement of plasma sample, while inspection
Survey its tissue samples, plasma sample and hydrothorax sample.
Hydrothorax sample is using the extraction in embodiment 1 and embodiment 2 and library constructing method；Tissue samples and hydrothorax are precipitated
The extraction of the DNA sample in sample uses conventional extraction process, and the extraction of cfDNA uses DNA extraction kit in plasma sample
Qiaamp Circulating Nucleic Acid Kit（QIAGEN）.
Testing result is as shown in table 6 below, and the wherein peculiar mutation recall rate of hydrothorax supernatant sample is similar with tissue samples, is better than
Plasma sample and hydrothorax precipitation sample；Hydrothorax supernatant sample is equal compared with the mutation recall rate of plasma sample/hydrothorax precipitation sample
Reach the level of signifiance（Blood plasma vs hydrothorax supernatants, p=0.02；Hydrothorax supernatant vs hydrothorax is precipitated, p=0.02）.
The peculiar mutation recall rate of the tumour of table 10 compares
Secondly, wherein 24 patients for having passed through traditional technique in measuring are contrasted with NGS detections, important driving gene
Detection situation, for EGFR, ALK gene exist mutation testing result it is as follows.In terms of sensitiveness, based on NGS methods, chest
Clear sensitiveness waterborne precipitates sample and plasma sample higher than hydrothorax.
The hydrothorax sample of table 11 and plasma sample detection EGFR 19 Exon deletion Sensitivity and Specificity contrasts
The hydrothorax sample of table 12 and plasma sample detection EGFR L858R Sensitivity and Specificity contrasts
The hydrothorax sample of table 13 and plasma sample detection ALK fusion Sensitivity and Specificity contrasts
In summary it can be seen, the peculiar mutation recall rates of hydrothorax supernatant extraction cfDNA are approximate with tissue samples, and sensitiveness is better than blood plasma
Sample and hydrothorax precipitation sample, and the important detection hydrothorax supernatant sample for driving gene also shows that big advantage, therefore
Hydrothorax supernatant sample can be extensive in Non-invasive detection aspect possible application as effective supplement of plasma sample.
Claims (10)
1. it is a kind of from hydrothorax extract cfDNA method, it is characterised in that comprise the following steps：
1st step, is centrifuged to hydrothorax sample；
2nd step, adds Carrier RNA, Proteinase K, lysate in the supernatant that the 1st step is obtained, and carries out incubation treatment, then add
Enter DNA and separate out liquid；
3rd step, the sample that the 2nd step is obtained is adsorbed by the way of post absorption to cfDNA, then is passed through after desorption, is obtained
Get cfDNA.
2. it is according to claim 1 from hydrothorax extract cfDNA method, it is characterised in that in described lysate wrap
Contain 100 mmol Tris-HCl, 25 mmol EDTA, 500 mmol NaCl, 1% SDS；Described DNA is included in separating out liquid
There are 0.15 mol/L NaCl, 0.02~0.05 M phosphate solutions；Pellosil adsorption column is used in post absorption.
3. it is according to claim 1 from hydrothorax extract cfDNA method, it is characterised in that before being desorbed, according to
Secondary use salt wash solution and alcohol wash solution are rinsed to adsorption column；Included in described salt wash solution：10~20
MM trishydroxymethylaminomethanes；1~5 M guanidine hydrochlorides, 10~30 mM EDTA-2Na, 0.1~1% HCl；Described alcohol washing
Solution is by the ethanol and isopropanol 1 of 75~80vol.%:1 prepares；The addition of described Carrier RNA is every milliliter
5~50 μ l are added in hydrothorax sample, the addition of Proteinase K is 50~500 μ l of addition in every milliliter of hydrothorax sample, lysate
Addition is 0.5~5ml of addition in every milliliter of hydrothorax sample, and the addition that DNA separates out liquid is addition in every milliliter of hydrothorax sample
0.5~5ml；The consumption of salt wash solution is every milliliter of 30~100 μ l of hydrothorax sample correspondence；The consumption of alcohol wash solution is every milli
Rise 150~300 μ l of hydrothorax sample correspondence；The used stripping liquid of desorption is without enzyme water.
4. it is a kind of from hydrothorax extract cfDNA kit, it is characterised in that include：Lysate, DNA separate out liquid, protease
K, Carrier RNA, adsorption column, salt wash solution, alcohol wash solution, stripping liquid.
5. it is according to claim 4 from hydrothorax extract cfDNA kit, it is characterised in that lysate, DNA separate out
Liquid, Proteinase K, Carrier RNA, salt wash solution, the volume range of alcohol wash solution are：500~5000：500~
5000：50~500：5~50：30~100：150~300；Described desorbed solution is without enzyme water.
6. the cfDNA for obtaining is extracted from hydrothorax and builds the cfDNA libraries for obtaining.
7. application of the cfDNA libraries described in claim 6 in gene sequencing.
8. application according to claim 7, it is characterised in that described gene sequencing is using NGS methods.
9. a kind of construction method in cfDNA libraries, it is characterised in that comprise the following steps：
1st step, size fragment separation is carried out to extracting the cfDNA for obtaining from hydrothorax；
2nd step, end reparation, 3' ends is carried out to small pieces cfDNA and, plus joint, purifying, is obtained plus base A, two ends successively
To cfDNA libraries；Size fragment in the 1st described step is separated to be separated using paramagnetic particle method.
10. a kind of cfDNA library construction Kits and its application in gene sequencing, claim is contained in the kit
The kit of the extraction cfDNA described in 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710117869.8A CN106867996B (en) | 2017-03-01 | 2017-03-01 | A kind of cfDNA libraries of the method that cfDNA is extracted from hydrothorax, kit and structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710117869.8A CN106867996B (en) | 2017-03-01 | 2017-03-01 | A kind of cfDNA libraries of the method that cfDNA is extracted from hydrothorax, kit and structure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106867996A true CN106867996A (en) | 2017-06-20 |
| CN106867996B CN106867996B (en) | 2018-05-18 |
Family
ID=59169194
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710117869.8A Active CN106867996B (en) | 2017-03-01 | 2017-03-01 | A kind of cfDNA libraries of the method that cfDNA is extracted from hydrothorax, kit and structure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106867996B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315393A (en) * | 2018-01-26 | 2018-07-24 | 中国医学科学院放射医学研究所 | The quantitatively method of detection dissociative DNA, application and the kit for detecting dissociative DNA |
| CN108893525A (en) * | 2018-07-12 | 2018-11-27 | 武汉拜施普生物科技有限公司 | A kind of blood circulation Tumour DNA new method for extracting |
| WO2019067973A1 (en) * | 2017-09-28 | 2019-04-04 | Grail, Inc. | Enrichment of short nucleic acid fragments in sequencing library preparation |
| CN109652513A (en) * | 2019-02-25 | 2019-04-19 | 元码基因科技（北京）股份有限公司 | The method and kit of liquid biopsy idiovariation are accurately detected based on two generation sequencing technologies |
| CN110129314A (en) * | 2019-04-24 | 2019-08-16 | 合肥欧创基因生物科技有限公司 | A kind of buffered with amino acid liquid and plasma DNA extracts kit |
| WO2019223502A1 (en) * | 2018-05-20 | 2019-11-28 | 江苏宏微特斯医药科技有限公司 | Method for detecting pathogens based on cfdna high-throughput sequencing |
| CN113403395A (en) * | 2021-06-03 | 2021-09-17 | 南京世和基因生物技术股份有限公司 | Method and kit for extracting cfDNA of aqueous humor and application of kit in PVRL clinical auxiliary examination |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5654179A (en) * | 1990-11-14 | 1997-08-05 | Hri Research, Inc. | Nucleic acid preparation methods |
| CN103131779A (en) * | 2013-02-25 | 2013-06-05 | 深圳市宝安区人民医院 | Enzyme digestion enriched PCR method used in noninvasive detection of epidermal growth factor receptor T790M mutation |
| CN103572378B (en) * | 2013-10-28 | 2015-12-02 | 博奥生物集团有限公司 | Based on Ion Proton tMthe construction process of the small segment DNA library of order-checking platform and application thereof |
-
2017
- 2017-03-01 CN CN201710117869.8A patent/CN106867996B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5654179A (en) * | 1990-11-14 | 1997-08-05 | Hri Research, Inc. | Nucleic acid preparation methods |
| CN103131779A (en) * | 2013-02-25 | 2013-06-05 | 深圳市宝安区人民医院 | Enzyme digestion enriched PCR method used in noninvasive detection of epidermal growth factor receptor T790M mutation |
| CN103572378B (en) * | 2013-10-28 | 2015-12-02 | 博奥生物集团有限公司 | Based on Ion Proton tMthe construction process of the small segment DNA library of order-checking platform and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GUO JIAN等: "Prediction of epidermal growth factor receptor mutations in the plasma/pleural eVusion to eYcacy of geWtinib treatment in advanced non-small cell lung cancer", 《J CANCER RES CLIN ONCOL》 * |
| MAGEN: "组织/血液D N A高通量提取", 《HIPURE DNA 96 KITS》 * |
| XIAOZHU ZHANG等: "Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer", 《LUNG CANCER》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019067973A1 (en) * | 2017-09-28 | 2019-04-04 | Grail, Inc. | Enrichment of short nucleic acid fragments in sequencing library preparation |
| EP4026915A1 (en) * | 2017-09-28 | 2022-07-13 | Grail, LLC | Enrichment of short nucleic acid fragments in sequencing library preparation |
| US11851650B2 (en) | 2017-09-28 | 2023-12-26 | Grail, Llc | Enrichment of short nucleic acid fragments in sequencing library preparation |
| EP4269583A3 (en) * | 2017-09-28 | 2024-01-17 | Grail, LLC | Enrichment of short nucleic acid fragments in sequencing library preparation |
| CN108315393A (en) * | 2018-01-26 | 2018-07-24 | 中国医学科学院放射医学研究所 | The quantitatively method of detection dissociative DNA, application and the kit for detecting dissociative DNA |
| WO2019223502A1 (en) * | 2018-05-20 | 2019-11-28 | 江苏宏微特斯医药科技有限公司 | Method for detecting pathogens based on cfdna high-throughput sequencing |
| CN108893525A (en) * | 2018-07-12 | 2018-11-27 | 武汉拜施普生物科技有限公司 | A kind of blood circulation Tumour DNA new method for extracting |
| CN109652513A (en) * | 2019-02-25 | 2019-04-19 | 元码基因科技（北京）股份有限公司 | The method and kit of liquid biopsy idiovariation are accurately detected based on two generation sequencing technologies |
| CN109652513B (en) * | 2019-02-25 | 2022-08-23 | 元码基因科技（北京）股份有限公司 | Method and kit for accurately detecting individual mutation of liquid biopsy based on second-generation sequencing technology |
| CN110129314A (en) * | 2019-04-24 | 2019-08-16 | 合肥欧创基因生物科技有限公司 | A kind of buffered with amino acid liquid and plasma DNA extracts kit |
| CN113403395A (en) * | 2021-06-03 | 2021-09-17 | 南京世和基因生物技术股份有限公司 | Method and kit for extracting cfDNA of aqueous humor and application of kit in PVRL clinical auxiliary examination |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106867996B (en) | 2018-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106867996B (en) | A kind of cfDNA libraries of the method that cfDNA is extracted from hydrothorax, kit and structure | |
| Freitas et al. | The role of liquid biopsy in early diagnosis of lung cancer | |
| CN107723352A (en) | A kind of Circulating tumor DNA liver cancer drives gene high-flux detection method | |
| CN109371018B (en) | Reinforcing agent for extracting free circulating tumor DNA, kit and method for extracting free circulating tumor DNA in peripheral blood | |
| US20190225958A1 (en) | Method for enrichment and purification of cell-free dna from body fluid for high-throughput processing | |
| CN104024426B (en) | The method for tracing of testing sample and detection kit in the secondary sequencing technologies of DNA | |
| CN112410327A (en) | Kit and method for extracting RNA | |
| CN106967712A (en) | Faeces DNA rapid extraction kit | |
| CN107541561A (en) | Improve kit, the device and method of fetus dissociative DNA concentration in maternal peripheral blood | |
| CN109161543B (en) | DNA probe for enriching low-frequency DNA mutation and application thereof | |
| CN111154872A (en) | Probe and kit for detecting lung cancer driving gene mutation | |
| Wu et al. | Extracellular RNA as a kind of communication molecule and emerging cancer biomarker | |
| CN107075732B (en) | Method for constructing free DNA sequencing library | |
| CN106282361B (en) | Gene capturing kit for capturing genes related to blood diseases | |
| EP3995588A1 (en) | Library creation method and application | |
| CN103789197B (en) | A kind of test kit and extracting method thereof extracting Microrna | |
| Trier Maansson et al. | Cell‐free chromatin immunoprecipitation can determine tumor gene expression in lung cancer patients | |
| CN108300774A (en) | The improved open chromatin detection method of one kind and its kit and application | |
| Li et al. | Whole-exome sequencing of rectal neuroendocrine tumors | |
| CN106702497B (en) | Kit for detecting free DNA in peripheral blood of pregnant woman and library building method | |
| CN106755505A (en) | Kit for detecting genetic mutation in blood plasma ctDNA | |
| CN108342488B (en) | Kit for detecting gastric cancer | |
| CN103667267A (en) | DNA (Deoxyribonucleic Acid) probe library used for hybridization with KRAS gene and method for enriching KRAS gene segments by adopting DNAprobe library | |
| CN109439655A (en) | The kit and method extracted suitable for ultramicron cellular nucleic acid | |
| CN114214408A (en) | Method, probe library and kit for detecting tumor ctDNA methylation with high throughput and high sensitivity |
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 | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Shao Yang Inventor after: Wang Xiaonan Inventor after: Wu Xue Inventor after: Wang Fufeng Inventor after: Bao Hua Inventor before: Shao Yang Inventor before: Wang Xiaonan Inventor before: Wu Xue Inventor before: Wang Fufeng Inventor before: Bao Hua Inventor before: Liu Yichuan |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20180628 Address after: 210032 A-7 building, -1 3, new Kumho Road, Jiangbei new district, Nanjing, Jiangsu. Patentee after: Nanjing and the medical equipment Co., Ltd. Address before: 210061, 18 building, 02 of the Ecological Life Science Industrial Park, 3-1 new Kumho Road, Nanjing hi tech Development Zone, Jiangsu. Patentee before: GENESEEQ TECHNOLOGY Inc. |