CN106867996B - A kind of cfDNA libraries of the method that cfDNA is extracted from hydrothorax, kit and structure - Google Patents

A kind of cfDNA libraries of the method that cfDNA is extracted from hydrothorax, kit and structure Download PDF

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CN106867996B
CN106867996B CN201710117869.8A CN201710117869A CN106867996B CN 106867996 B CN106867996 B CN 106867996B CN 201710117869 A CN201710117869 A CN 201710117869A CN 106867996 B CN106867996 B CN 106867996B
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cfdna
hydrothorax
sample
wash solution
dna
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CN106867996A (en
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邵阳
汪笑男
吴雪
王富锋
包华
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Nanjing and the medical equipment Co., Ltd.
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南京世和基因生物技术有限公司
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    • 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
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    • 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
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    • 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
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    • 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, for building the method for 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 be built as high-throughput sequencing library from hydrothorax for the first time, and can become one kind effectively supplement of blood plasma cfDNA；In addition, in this method by hydrothorax sample cfDNA sizes segment separation after, so that the cfDNA purity highers in hydrothorax sample, make its structure more suitable for 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

A kind of cfDNA of the method that cfDNA is extracted from hydrothorax, kit and structure Library
Technical field
The present invention relates to the cfDNA libraries of a kind of method that cfDNA is extracted from hydrothorax, kit and structure, belong to Technical field of gene detection.
Background technology
With being continuously increased for morbidity and mortality, cancer has become the first cause of Chinese population death and main Public health problem.It is counted according to National Cancer Center, China in 2015 is there are about 4,290,000 cancer new cases, 2,810,000 Cancer death, lung cancer become the first cause of most common cancer and cancer mortality.With various treatment means Constantly rise, such as the radiotherapy of early stage, the chemotherapy in modern age targeted therapy till now and immunization therapy, the treatment of cancer just gradually to Precision treatment mode develops, and is especially targeted treatment and immunization therapy, thus seems for the screening of selectively targeted crowd It is particularly important, and as clinical supplementary means indispensable in targeted drug therapy, the genetic test of cancer is directly related to Drug is for the therapeutic efficiency of patient.
High throughput sequencing technologies are also known as next-generation sequencing technologies, since it is high-throughput, the characteristics such as sensitivity height, in cancer It is used widely in genetic test.A ring mostly important as high-flux sequence, the preparation of library sample seem particularly heavy Will, current commonplace detection sample has the tumor tissues sample of patient, puncture 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 existing DNA that dissociates in blood plasma, cfDNA is damaged from body cell, and some comes from In normal cell, some comes from abnormal cell（Such as tumour cell）.For Healthy People, the cfDNA main sources in blood It is that body cell is normally metabolic, therefore a relatively low level should be maintained.However, working as has abnormal cell（Tumour is thin Born of the same parents）When being formed or having very serious systemic organ damage, the cell of mortality（Either apoptosis or necrosis）It can produce Raw substantial amounts of cfDNA.And its non-invasive characteristic is praised highly extensively, and 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, it is influenced by many factors, the shape residing for tumour The current stable disease of state such as patient still influences the detection of the peculiar mutation of tumour, such cancer staging into exhibition, and cancer turns Position of shifting etc. may all influence the detection of ctDNA in blood plasma, therefore there is an urgent need for a kind of supplements of liquid biopsy sample.
Hydrothorax is mainly late most commonly seen in lung cancer, and 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 It invades and blocks caused by capillary and lymphatic vessel.
The content of the invention
It is explored by a large number of experiments, the present invention has unexpectedly discovered that a kind of can be extracted 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 sufficient；And after handling sample, the structure of DNA library, this method extraction can be effectively realized Obtained cfDNA by high-flux sequence, can effectively detect the mutation of patient's carrying, and it was found that in this way After obtained cfDNA structures library sequencing, its recall rate being mutated is higher than in blood/plasma sample in some samples CfDNA, so as to more can effectively prompt the information such as patient's prognosis, medication.
The first aspect of the invention：
A kind of method that cfDNA is extracted from hydrothorax is provided, is included the following steps：
Hydrothorax sample is centrifuged in 1st step；
2nd step adds in Carrier RNA, Proteinase K, lysate in the supernatant obtained in the 1st step, carries out incubation processing, It adds DNA and liquid is precipitated；
3rd step is adsorbed cfDNA the samples that the 2nd step obtains by the way of column absorption, using desorption Afterwards, cfDNA is got.
In one embodiment, 50~150 mmol Tris-HCl, 20~30 mmol are included in the lysate EDTA, 400~600 mmol NaCl, 0.5~1.5% SDS.
In one embodiment, the DNA, which is precipitated in liquid, includes 0.1~0.2 mol/L NaCl, 0.02~0.05 M phosphate solutions.
In one embodiment, using pellosil adsorption column in column absorption.
In one embodiment, before being desorbed, successively using salt wash solution and alcohol wash solution to adsorption column It is rinsed.
Include in the salt wash solution：10~20 mM trishydroxymethylaminomethanes, 1~5 M guanidine hydrochlorides, 10 ~30 mM EDTA-2Na, 0.1~1% HCl.
The alcohol wash solution is by the ethyl alcohol of 75~80vol.% and isopropanol 1:1 prepares.
The addition of the Carrier RNA is 5~50 μ l of addition in every milliliter of hydrothorax sample, and Proteinase K adds It is 50~500 μ l of addition in every milliliter of hydrothorax sample to enter amount, the addition of lysate be add in 0.5 in every milliliter of hydrothorax sample~ The addition that liquid is precipitated in 5ml, DNA is 0.5~5ml of addition in every milliliter of hydrothorax sample；The dosage of salt wash solution is every milliliter Hydrothorax sample corresponds to 30~100 μ l；The dosage of alcohol wash solution is that every milliliter of hydrothorax sample corresponds to 150~300 μ l.
Stripping liquid is no enzyme water used by desorption.
The second aspect of the invention：
A kind of kit that cfDNA is extracted from hydrothorax is provided, is included：Lysate, DNA be precipitated liquid, Proteinase K, Carrier RNA, adsorption column, salt wash solution, alcohol wash solution, stripping liquid.
Liquid, Proteinase K, Carrier RNA, salt wash solution, the volume ratio model of alcohol wash solution is precipitated in lysate, DNA Enclosing is：500~5000：500~5000：50~500：5~50：30~100：150~300.
The stripping liquid is no enzyme water.
The third aspect of the invention：
It provides and is built to obtain cfDNA libraries by the cfDNA extracted from hydrothorax.
The fourth aspect of the invention：
Provide application of the above-mentioned cfDNA libraries in gene sequencing.
In one embodiment, the gene sequencing is using NGS（Next generation's sequencing）Method.
The fifth aspect of the invention：
A kind of construction method in cfDNA libraries is provided, is included the following steps：
1st step carries out size segment separation to the cfDNA that extraction obtains；
2nd step carries out small pieces cfDNA end reparation successively, 3' ends add base A, both ends add connector, pure Change, obtain cfDNA libraries.
In one embodiment, the size segment separation in the 1st step 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, in the kit Contain the kit of said extracted cfDNA.
Advantageous effect
The present invention has successfully extracted cfDNA from hydrothorax for the first time, and empirical tests can be used as high-throughput sequencing library to build Samples sources, and can as blood plasma cfDNA one kind effectively supplement；In addition, by hydrothorax sample in this method After the separation of cfDNA sizes segment so that the cfNDA purity highers in hydrothorax sample make its structure more suitable for 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.
Description of the drawings
Fig. 1 is that hydrothorax supernatant cfDNA passes through the unsegregated Quality Control figure of size segment；
Fig. 2 is Quality Control figures of the hydrothorax supernatant cfDNA by size segment after separation；
Fig. 3 is the Quality Control figure in the cfDNA structures library obtained in embodiment 2 by Beads enrichment；
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.Specific skill is not specified in embodiment Art or condition person carry out according to the described technology of document in the art or condition or according to product description.Examination used Production firm person is not specified in agent or instrument, and being can be with conventional products that are commercially available.Word " comprising " used herein, "comprising", " having " or its any other variant are intended to cover including for non-exclusionism.E.g., including list element technique, Method, article or equipment are not necessarily limited by those elements, but can be not explicitly listed including other or belong to this work Skill, method, article or the intrinsic element of equipment.Unless the context clearly dictates, 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 no special instruction Than.
" hydrothorax sample " described in following embodiment refers to original acquirement hydrothorax sample in the case of no special instruction Originally, it is using initial hydrothorax sample volume as standard stabiliser with the dosage of reagent in extraction process.
It has passed through a large number of experiments to grope, the present invention has unexpectedly discovered 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 mainly passes through centrifugation, egg White enzyme K processing, the method for column absorption obtain the cfDNA in hydrothorax.
The extraction of 1.cfDNA
1.1 it centrifuges：
In centrifugation step, main purpose is to make the separation such as cell, high molecular weight protein, blood formed element in hydrothorax, Supernatant is obtained, present invention finds cfDNA is contained in supernatant, these cfDNA are suitable for library construction after extraction, can There is higher mutation recall rate than the cfDNA that blood sample obtains.Here centrifugation step, can be in 1000~20000 Rpm centrifuges 5~30 min, and supernatant is in 4 DEG C of preservations.
The processing of 1.2 protein breakdowns：
Addition Carrier RNA are also needed in hydrothorax supernatant can help nucleic acid to remove electrostatic effect, can be adsorbed to very well On purification column, elution efficiency can be improved, so as to reach recycling sample amplifying nucleic acid as big as possible, is next needed by protease Resolution process, using Proteinase K to sample incubation processing, Proteinase K can decompose the protein in supernatant, be incubated process Temperature can be controlled in 45 DEG C of water-bath 10min；After treatment needs in sample to add in lysate, the lysate used here Ingredient can be comprising 50~150 mmol Tris-HCl, 20~30 mmol EDTA, 400~600 mmol NaCl, 0.5 ~1.5% SDS；After incubation, it is also necessary to add DNA and liquid is precipitated to maintain sample system, a less salt ring can be provided Border is conducive to the precipitation of dissociative DNA, in addition, promoting the salt of residual protein molten by less salt, and removes residual protein, subtracts The pollution of few subsequent protein, the DNA, which is precipitated in liquid, includes 0.1~0.2 mol/L NaCl, 0.02~0.05 M phosphorus Acid salt solution.
1.3 columns adsorb：
After Proteinase K is obtained treated sample, column adsorption operations are ready for, present invention discover that being inhaled by this column Subsidiary formula method can obtain the cfDNA of more high quality compared with other methods, and silicon may be employed in adsorption column adopted here Glued membrane column after adsorption process, is rinsed using cleaning solution, can effectively get rid of the impurity in sample and retain compared with More cfDNA in a preferred embodiment, may be employed two kinds of lavation buffer solutions and be rinsed successively, the composition of salt wash solution It is：10~20 mM trishydroxymethylaminomethanes, 1~5 M guanidine hydrochlorides, 10~30 mM EDTA-2Na, 0.1~1% HCl, alcohol The composition of wash solution is：The ethyl alcohol of 75~80vol.% and isopropanol 1:1 compounding.
After impurity is washed, the cfDNA of absorption is eluted using stripping liquid, obtains the elution containing cfDNA Liquid, eluent here is preferably using no enzyme water.
2. library construction：
After obtaining cfDNA, after being built library, mutation recall rate more higher than plasma sample can be shown.This In structure library method, can include and size segment separation is carried out to the obtained cfDNA of extraction；To small pieces cfDNA Successively carry out end reparation, 3' ends plus base A, both ends plus connector, purifying and etc..
2.1 Beads enrichment
Wherein, it is committed step to carry out the separation of size segment to the cfDNA that extraction obtains, by hydrothorax sample After the separation of cfDNA sizes segment so that the cfNDA purity highers in hydrothorax sample avoid big segment cfDNA for flux It occupies, makes its structure more suitable for DNA library, be more conducive to the detection of later stage mutation, the abundance meeting higher of detection also more may be used Letter can solve the problems, such as the conventional sample unicity and recall rate that storehouse is only built by blood plasma extraction cfDNA.Here size segment Separation the mode of Beads enrichment may be employed, by being adsorbed to cfDNA, then after large fragment is washed, by small fragment cfDNA from It is eluted on magnetic bead.
It repairs 2.2 ends
CfDNA segments are carried out end reparation can utilize Klenow segments, T4DNA polymerases and T4 polynucleotide kinases It carries out, wherein, the Klenow segments have 5 ' -3 ' ' polymerase activities and 3 ' -5 ' polymerase activities, but it is circumscribed to lack 5 ' -3 ' Enzymatic activity.Thereby, it is possible to easily and accurately carry out end reparation to cfDNA segments.It according to an embodiment of the invention, can also be into The step of one step includes purifying the cfDNA segments repaired by end, thus, it is possible to easily carry out subsequent processing.
Using T4 polymerases and Klenow Escherichia coli polymerase segments, for cfDNA 5' protrude viscous end-filling and 3' protrudes viscous end and ties, and generates flat end, is connected for subsequent flush end.Reaction carries 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 segments repaired by end, to obtain with cohesive end A's CfDNA segments.According to one embodiment of present invention, Klenow (3 ' -5 ' exo-) can be utilized, i.e., with 3 ' -5 ' excision enzymes The Klenow of activity, in 3 ' ends of the cfDNA segments repaired by end addition base A.Thereby, it is possible to easily and accurately will Base A is added to 3 ' ends of the cfDNA segments repaired by end.According to an embodiment of the invention, can also further wrap The step of being purified to the DNA fragmentation with cohesive end A is included, thus, it is possible to easily carry out subsequent processing.
Reaction carries out in PCR amplification instrument, 37 DEG C, 30 minutes.
Table 2
CDNA/DNA crosses column purification, commercialization company purification kit.
2.4cfDNA both ends add connector
Table 3
2.5 DNA amplification templates
In one embodiment of the invention, cfDNA fragment amplifications enable the amount of nucleic acid to meet chip/probe hybridization The demand of capture.
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 pre-degenerations 30 seconds, 98 DEG C are denatured 30 seconds, and 65 DEG C are annealed 30 seconds, 72 DEG C Extension 30 seconds cycles 4-6 times altogether.Finally extend 5 minutes at 72 DEG C.
The cfDNA libraries that the above method obtains can be applied in non-treatment and the gene sequencing of diagnostic purpose, particularly It is sequenced suitable for 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 1 hydrothorax sample of embodiment
The separation of 1 hydrothorax sample supernatant precipitation
The hydrothorax sample of fresh acquisition is taken, 1800rpm centrifuges 10 min, hydrothorax supernatant is adsorbed in new centrifuge tube, Hydrothorax precipitation is discarded, by hydrothorax supernatant Sample preservation to 4 DEG C（-80℃）Refrigerator.
The column adsorbing separation of cfDNA in 2 hydrothorax supernatants
2.1 sampling：Take out 4 DEG C（-80℃）The hydrothorax supernatant sample of preservation, at 4 DEG C of 10 min of 16000rpm Reason；
2.2 configuration premixed liquids：Prepare lysate Buffer ACL Mix（QIAGEN）, 2 times of hydrothorax total volumes of absorption Buffer ACL Mix overturn mixing；
2.3 add Proteinase K：20 μ l Carrier RNA are added in every milliliter of hydrothorax sample, add 200 μ l protease K fully digests；
2.4 add in ACL：By adding in 2 ml lysate Buffer ACL Mix in every milliliter of hydrothorax sample（QIAGEN）, 45 DEG C water-bath 10min, incubation terminate, room temperature cooling；
2.5 add in ACB：Liquid Buffer ACB are precipitated by 2ml DNA are added in every milliliter of hydrothorax sample（QIAGEN）, top Mixing；
2.6 prepare to take out pump：Rule plug is removed, QIAamp Vac is plugged, plugs connexon, loads onto pillar, keeps lid Closed state；
2.7 cross column：50ml mixed liquors are transferred in sample tube, vacuum pump is opened, at the uniform velocity passes through pellosil adsorption column （QIAGEN）；
2.8 elution：300 μ l salt wash solution Buffer ACW1 are added in adsorption column（QIAGEN）, vacuum pump is opened, Slowly evenly by adsorption column, 1000 μ l alcohol wash solution Buffer ACW2 are continuously added（QIAGEN）；Continue to adsorption column It adds in 450 μ l absolute ethyl alcohols to elutions to terminate, closes vacuum pump；
2.9 samples centrifuge：Adsorption column lid is covered, is placed in centrifuge tube, 3000rpm centrifugations 3min；2.10 recycling QIAamp vac plug Rule plug；
2.11 eluted dna：5-10 μ l are added in without enzyme water to adsorption column, and 1800rpm centrifugation 30s turn the DNA after elution It moves on in new 1.5ml adsorption tubes, adds in 30 μ L without enzyme water, stand 3min, 2500rpm centrifugation 2.12 DNA mixings of 5min：With Vortex vortex mixings.
3 cfDNA samples quantify
By the DNA sample after mixing, 1 μ L is taken to carry out Qubit and are quantified.
1 phenol-chloroform of reference examples-isoamyl alcohol extraction method extraction cfDNA
1. hydrothorax 4ml, 2000rpm room temperature is taken to centrifuge 10 min, supernatant is moved in new centrifuge tube, then through 12000rpm After room temperature centrifugation 5min, supernatant is moved in new centrifuge tube.
2. 20 μ l of 1%SDS solution 350ml, 20mg/ml Proteinase K Solution will be added in supernatant, whirlpool concussion mixing, 58 DEG C 75 min of water-bath adds in 700 μ l of phenol-chloroform-isoamyl alcohol mixed liquor, and whirlpool shakes mixing, and 12000rpm centrifuges 10min at 4 DEG C, Supernatant is moved in new centrifuge tube, addition 800 μ l of isopropanol, reverse mixing, 12000rpm centrifugation 10min at 4 DEG C, in tipping Clearly, 75% ethyl alcohol 1ml is added in, overturns mixing, 12000rpm centrifuges 10min at 4 DEG C, and tipping supernatant, 65 DEG C of metal baths are until remaining Liquid is completely dried, and adds in 10 μ l of deionized water, 65 DEG C of dissolving 10min, -20 DEG C preserve.
2 phenol-chloroforms of reference examples-isoamyl alcohol extraction cooperation minim DNA settling agent method extraction cfDNA
Difference with reference examples 1 is：5 μ l of minim DNA settling agent are added in while 800 μ l of isopropanol are added in.
Reference examples 3
Difference with embodiment 1 is：The addition volume of first buffer solution is added in hydrothorax supernatant sample equivalent.
Reference examples 4
Difference with embodiment 1 is：DNA is not added in, and liquid is precipitated.
Using the DNA sample obtained in above-described embodiment 1 and reference examples 1~4,1 μ l is taken to carry out Qubit and are quantified.
5 DNA sample quantitative result of table
As can be seen from the above table, method using the present invention can obtain the cfDNA samples of high-content from hydrothorax, excellent In conventional method and reference examples method, the DNA that do not add in wherein in reference examples 4 is precipitated liquid processing, can not only improve DNA and contain Amount also makes DNA mass be improved, thus it is speculated that it, which is acted on, includes 1, protein is precipitated；2nd, the remaining protein of removal, makes extinction Rate ratio A260/A280 meets the requirements between 1.8 to 2.
The structure of 2 DNA library of embodiment
The separation of 3.1 cfDNA size segments
A) embodiment 1 is extracted cfDNA samples adds no enzyme water polishing to add in 35 μ l magnetic beads Axygen to 50 μ l Beads, with liquid-transfering gun mixing；
B) sample is placed on magnetic separation frame until supernatant is clarified, and absorption supernatant is into another 1.5ml low adsorptions pipe；
C) add in 65 μ l magnetic bead Axygen beads resuspensions liquid-transfering gun mixing and be placed on magnetic separation frame up to upper limpid Clearly, supernatant is abandoned, adds in the cleaning of 50 μ l, 75% ethyl alcohol, liquid-transfering gun is removed raffinate, dried；
D) plus 56 μ L are without enzyme water, and mixing pipettes 51 μ l clarified solutions into new 1.5ml centrifuge tubes；
e）1 μ l is taken to measure DNA concentration with Qubit.50 μ l ctDNA small fragments of gained are directly used in cfDNA KAPA and build storehouse Flow.
Magnetic bead size segment separates front and rear cfDNA Quality Controls figure as depicted in figs. 1 and 2, it can be seen from the figure that by magnetic After pearl separation, size segment has obtained preferable separation, and the cfDNA contents of small-molecular-weight significantly improve.
3.2 cfDNA duplex ends reparations
50 μ L cfDNA after constant volume are all added in the reaction systems of tables 6, be placed on after mixing in PCR instrument 20 DEG C it is anti- Answer 30min, 65 DEG C of reaction 30min.
Table 6cfDNA duplex ends repair reaction system
3.3 Adaptor are linked
A) connector coupled reaction is configured
7 connector coupled reaction reaction system of table
b）Brief centrifugation and with the abundant mixings of p200.20 DEG C in PCR instrument, 60min.
3.4 product purification
A) 50 μ L are added in and are resuspended Axygen beads, liquid-transfering gun mixing, 5min on magnetic separation frame until supernatant is clarified, Supernatant is abandoned, 50 μ L, 75% ethyl alcohol is added to wash 2 times, removes residul liquid-removing；
E) plus 30 μ L are without enzyme water, liquid-transfering gun mixing, 5min on magnetic frame；
g）1 μ L is taken to carry out Qubit to quantify, remaining enters following PCR together with beads
3.5 PCR amplification enriched products
The supernatant that above-mentioned steps are obtained adds in amplification system, and specific formula is shown in Table 8, after mixing, by reaction system It 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 purification
A) add in 25 μ L magnetic bead Axygen beads in above-mentioned product to be resuspended, liquid-transfering gun mixing, it is supreme on magnetic frame It is limpid clear, supernatant is abandoned, the ethyl alcohol for adding in 50 μ L 75% cleans 2 times, and room temperature dries 5min；
b）Adding 31 μ L, liquid-transfering gun mixing is incubated at room temperature 20min, clarified solution is moved to the low suctions of new 1.5ml without enzyme water In attached EP pipes.
c）1 μ L is taken to carry out survey Qubit to quantify, remaining sample enters follow-up process as sequencing library.
The Quality Control figure in obtained library is as shown in Figure 3.As can be seen that the cfDNA structures obtained using extracting method of the present invention Library is built with better base quality.
It is mutated recall rate experiment
In order to probe into whether hydrothorax sample can randomly select 34 patients with lung cancer, together as the supplement of plasma sample When detect its tissue samples, plasma sample and hydrothorax sample.
Hydrothorax sample is using embodiment 1 and the extraction in embodiment 2 and library constructing method；Tissue samples and hydrothorax precipitation The extraction of DNA sample in sample using conventional extraction process, in plasma sample the extraction of cfDNA use DNA extraction kit 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 precipitates, p=0.02）.
The peculiar mutation recall rate of 10 tumour of table compares
Secondly, wherein 24 have been compared by the patient and NGS detections of traditional technique in measuring, important driving The detection situation of gene, the testing result that there is mutation for EGFR, ALK gene are as follows.In terms of sensibility, based on NGS side Method, the sensibility of hydrothorax supernatant is higher than hydrothorax precipitation sample and plasma sample.
11 hydrothorax sample of table and plasma sample detection EGFR 19 Exon deletion sensibility and specificity comparisons
12 hydrothorax sample of table and plasma sample detection EGFR L858R sensibility and specificity comparisons
13 hydrothorax sample of table and plasma sample detection ALK fusion sensibility and specificity comparisons
It in summary it can be seen, the peculiar mutation recall rates of hydrothorax supernatant extraction cfDNA are approximate with tissue samples, and sensibility is better than Plasma sample and hydrothorax precipitation sample, and the detection hydrothorax supernatant sample of important driving gene also shows that big advantage, Therefore hydrothorax supernatant sample can be extensive in Non-invasive detection level possible application as effective supplement of plasma sample.

Claims (4)

  1. A kind of 1. method that cfDNA is extracted from hydrothorax, which is characterized in that include the following steps：
    Hydrothorax sample is centrifuged in 1st step；
    2nd step adds in Carrier RNA, Proteinase K, lysate in the supernatant obtained in the 1st step, carries out incubation processing, then adds Enter DNA and liquid is precipitated；
    The sample that 2nd step obtains adsorbs cfDNA by the way of column absorption, after desorption, obtains by the 3rd step Get cfDNA；
    Include 100 mmol Tris-HCl, 25 mmol EDTA, 500 mmol NaCl, 1% SDS in the lysate；
    The DNA, which is precipitated in liquid, includes 0.15 mol/L NaCl, 0.02~0.05 M phosphate solutions；
    Using pellosil adsorption column in column absorption；
    Before being desorbed, adsorption column is rinsed using salt wash solution and alcohol wash solution successively；
    Include in the salt wash solution：10~20 mM trishydroxymethylaminomethanes；1~5 M guanidine hydrochlorides, 10~30 MM EDTA-2Na, 0.1~1% HCl；
    The alcohol wash solution is by the ethyl alcohol of 75~80vol.% and isopropanol 1:1 prepares；
    The addition of the Carrier RNA is 5~50 μ l of addition, the addition of Proteinase K in every milliliter of hydrothorax sample It is 50~500 μ l of addition in every milliliter of hydrothorax sample, the addition of lysate is 0.5~5ml of addition in every milliliter of hydrothorax sample, The addition that liquid is precipitated in DNA is 0.5~5ml of addition in every milliliter of hydrothorax sample；
    The dosage of salt wash solution is that every milliliter of hydrothorax sample corresponds to 30~100 μ l；
    The dosage of alcohol wash solution is that every milliliter of hydrothorax sample corresponds to 150~300 μ l；
    Stripping liquid is no enzyme water used by desorption；
  2. 2. a kind of kit that cfDNA is extracted from hydrothorax, which is characterized in that include：Liquid, protease is precipitated in lysate, DNA K, Carrier RNA, adsorption column, salt wash solution, alcohol wash solution, stripping liquid；Lysate, DNA be precipitated liquid, Proteinase K, Carrier RNA, salt wash solution, the volume ratio range of alcohol wash solution are：500~5000：500~5000：50~500：5 ~50：30~100：150~300；The desorbed solution is no enzyme water；
    Include 100 mmol Tris-HCl, 25 mmol EDTA, 500 mmol NaCl, 1% SDS in the lysate；
    The DNA, which is precipitated in liquid, includes 0.15 mol/L NaCl, 0.02~0.05 M phosphate solutions；
    Using pellosil adsorption column in column absorption；
    Include in the salt wash solution：10~20 mM trishydroxymethylaminomethanes；1~5 M guanidine hydrochlorides, 10~30 MM EDTA-2Na, 0.1~1% HCl；
    The alcohol wash solution is by the ethyl alcohol of 75~80vol.% and isopropanol 1:1 prepares.
  3. 3. a kind of construction method in cfDNA libraries, which is characterized in that include the following steps：
    1st step carries out size segment point to the cfDNA that the method described in claim 1 that cfDNA is extracted from hydrothorax obtains From；
    2nd step carries out small pieces cfDNA end reparation successively, 3' ends, plus connector, purifying, are obtained plus base A, both ends To cfDNA libraries；Size segment separation in 1st step is separated using paramagnetic particle method.
  4. Application of the 4.cfDNA libraries in gene sequencing；The cfDNA libraries refer to through the cfDNA described in claim 3 What the construction method in library obtained；The gene sequencing is using NGS methods；The gene is EGFR gene or ALK Gene；The application refers to NGS gene mutation sensitivity Detections.
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Publication number Priority date Publication date Assignee Title
EP3688191B1 (en) * 2017-09-28 2022-03-02 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
CN108660199A (en) * 2018-05-20 2018-10-16 北京宏微特斯生物科技有限公司 A method of pathogen is detected based on cfDNA high-flux sequences
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
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
CN110129314B (en) * 2019-04-24 2021-04-13 合肥欧创基因生物科技有限公司 Amino acid buffer solution and plasma free DNA extraction kit
CN113403395B (en) * 2021-06-03 2023-03-24 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)

* Cited by examiner, † Cited by third party
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Title
Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer;Xiaozhu Zhang等;《Lung Cancer》;20081231;第176页右栏第3-4段 *
Prediction of epidermal growth factor receptor mutations in the plasma/pleural eVusion to eYcacy of geWtinib treatment in advanced non-small cell lung cancer;Guo Jian等;《J Cancer Res Clin Oncol》;20101231;全文 *
组织/血液D N A高通量提取;Magen;《HiPure DNA 96 Kits》;20101231;第2-7页 *

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