CN105950739A - Probe for detecting circulating tumor DNA (Deoxyribonucleic Acid) of human breast cancer and application of probe - Google Patents
Probe for detecting circulating tumor DNA (Deoxyribonucleic Acid) of human breast cancer and application of probe Download PDFInfo
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- 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
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a probe for detecting a circulating tumor DNA (Deoxyribonucleic Acid) of a human breast cancer and application of the probe, belonging to the field of diagnosis and prevention of diseases. The invention designs a breast cancer selector which consists of DNA segments in mutation regions of related genes of the breast cancer. The circulating tumor DNA (ctDNA) of a breast cancer patient is subjected to liquid-phase hybridization capture by using the probe designed by the selector; after high-throughput sequencing, specific cancer gene variation of a patient is found, and a tumor load is monitored. Compared with a conventional detection means, the probe and the application thereof disclosed by the invention have the characteristics of no invasiveness, high throughout, good sensitivity and specificity, and the like; the probe and the application thereof can be used for better screening and early-stage diagnosis of the human breast cancer and are applied to individualized treatment of cancer.
Description
Technical field
The present invention relates to a kind of method for diagnosing the illness, particularly to a kind of method for the detection of human breast carcinoma Circulating tumor DNA, belong to the diagnosis and treatment field of disease.
Background technology
Breast carcinoma is the malignant tumor that whole world women sickness rate is the highest, and the whole world there are about 1,700,000 women every year and suffers from breast cancer, and 52.2 die ten thousand deaths in breast carcinoma.The sickness rate of whole world breast carcinoma is different and different from living habit because of area surroundings, economic disparities.In general, the developed country such as West Europe, North America sickness rate is higher, and the most area in Asia, Latin America and Africa is Di Fa district.In terms of global range, the sickness rate of the annual breast carcinoma in the whole world rises with the amplitude of 0.2%～8%, and breast carcinoma is first cause of death of 40～55 years old women.From late 1970s, the morbidity of breast carcinoma occupies the first place of female malignant the most always.Though China belongs to low area of women with breast cancer in the world, but the sickness rate of breast carcinoma substantially increases in recent years.China main cities over 10 years breast cancer incidence increase 37%, mortality rate increases 38.9%, becomes the fastest-rising cancer of mortality rate in city, and age of onset is also in the trend of gradually rejuvenation.Especially with Shanghai, Beijing, Tianjin and coastal area be China's breast carcinoma hotspot, the first place of Yi Zhan China female malignant sickness rate.Although the sickness rate of external breast carcinoma remains high ,But mortality rate constantly declines, its reason not only has benefited from Protocols in Molecular Biology and the raising of comprehensive diagnosis and treatment standardized level of development in recent years, more has benefited from women with breast cancer examination and early examines the foundation of system.
Breast carcinoma inspection technique the most current has 6 kinds: include checking oneself mammary gland, clinical health check-up, mammary X-ray film making, Breast ultrasonography, mammary gland nuclear magnetic resonance check, nipple discharge inspection.Wherein, owing to mammary gland nuclear magnetic resonance check equipment requirements is high, price is higher, the longest, needs intravenous injection reinforcing agent, is therefore difficult to promote.Acupuncture cell carries out nipple discharge inspection, due to reasons such as complex operation step, recall rate are the lowest, general it is not recommended that apply in Mass screening.
The best period of breast self-exams is after menstrual onset the 7th～10 day, and now the impact of estrogen on breast is minimum, and mammary gland is in relative static conditions, easily finds pathological changes.But there is data to show, by this means, the early diagnosis probability of breast carcinoma to be improved and reduce case fatality rate, the most also only as a kind of method improving anti-cancer consciousness.
Mammography is the effective ways of universally acknowledged detection breast carcinoma of early stage, especially Digital Mammography, it is possible to the clear fine structure at all levels showing mammary gland, the finest calcification.And fine calcification is the even unique sign of important sign of x-ray Diagnosis of Breast cancer.Abroad begin to use mammary X-ray to do large-scale breast carcinoma examination from the sixties in 20th century.Mammary X-ray examination is high to more than 40 years old Asia women's accuracy.Cancer Hospital of Chinese Academy of Medical Sciences and the breast carcinoma examination carried out of consonance medical university all using x-ray as first-selected Clinical screening method.But mammary X-ray is poor to young fine and close mammary gland tissue penetration power, thus general it is not recommended that to less than 40 years old, carry out mammography without the women that clear and definite high-risk breast cancer factor or clinic health check-up are no abnormal.Mammary X-ray has been short of in terms of differentiating mammary gland capsule and solid tumour；Examination scope has certain limitation, often omits mammary gland edge and the pathological changes of far-end；Poor to the photographic effects of the flat breast that withers.So still suffering from disputing on as the method for breast carcinoma examination at China's application mammography in nonpalpable breast.
Breast ultrasonography, because objectivity is poor, needs to rely on the experience of ultrasonic doctor, and subjective, outside native land, guide is not recommended as main screening method.Can be as the supplementary inspection measure that the joint inspection measure of mammary X-ray examination or mammary X-ray screening results are 0 grade of person of BI-RADS.In view of Chinese's pathogenesis of breast carcinoma peak is more forward, premenopausal patients's ratio is high, and mammary gland is relatively compact, ultrasonic can be as the supplementary means of mammary gland examination.
It is considered that present stage is necessary to find the breast carcinoma detection methods being more suitable for China's economic structure and crowd, examines and early control the morning of raising breast carcinoma.The early discovery of breast carcinoma, early diagnosis and early treatment are the unique effectively approach of practicality reducing mortality rate, raising cure rate at present.The breast carcinoma of early stage patient of 90% can cure, and advanced breast cancer treatment after, survival period reach more than 5 years only account for 20%.But from the point of view of the situation of China, in the patient with breast cancer gone to see a doctor, early cancer ratio is too low.The early rate of examining that improves is to improve the most critical link of patient with breast cancer's survival rate, how early detection and prevention for breast carcinoma provide feasible measure and alarm signal, and provide foundation for early diagnosis, early treatment, prophylaxis of tumours morbidity and reduction death, it has also become the emphasis of modern medicine study.
In recent years, along with developing rapidly of Protocols in Molecular Biology, circulation dissociative DNA detection application in breast carcinoma diagnosis and treatment process becomes study hotspot.The DNA being present in blood with extracellular free form is referred to as circulating dissociative DNA.The circulation dissociative DNA in tumor patient in-vivo tumour source is also called Circulating tumor DNA.Further investigation to circulation dissociative DNA finds, Circulating tumor DNA proportion is relevant with the size of cancerous tissue and state, can reflect the feature of cancerous tissue DNA, is used for assessing tumor load.Detected the disease cause mutation of breast carcinoma in the past, and needed the tumor tissues of biopsy or excision, belong to invasive inspection.Circulating tumor DNA is because having the feature identical with tumor tissues, therefore detect Circulating tumor DNA is to supplement conventional diagnostic method, the patient excised especially for primary lesion, the Circulating tumor DNA in its blood plasma can be as efficacy determination, the reliability index of prognosis detection.And for the examination of tumor high-risk, the detection of circulation dissociative DNA is non-invasive with it, specificity and highly sensitive feature, there is great advantage.And along with susceptiveness and the specificity of detection method improve constantly, the detection of Circulating tumor DNA becomes more accurate.
The detection of breast carcinoma Circulating tumor DNA disclosure satisfy that this area is in the urgent need to setting up new breast carcinoma inspection and the demand of monitoring technology, and can be in early days objective, sensitive, special and the most stable Diagnosis of Breast cancer, and monitoring and evaluation tumor load, to carry out in time, treat targetedly, reduce medical treatment cost, save social resources.
Summary of the invention
The technical problem to be solved is to provide a kind of high specificity for the detection of human breast carcinoma Circulating tumor DNA, highly sensitive experimental technique, compared with conventional breast cancer diagnosis method, the features such as the method for the present invention has quickly, high flux, sensitive and specificity are good, can preferably carry out early diagnosis and the monitoring breast cancer tumour load of human breast carcinoma.
In order to reach object above, the technological means that the present invention uses is:
Design a kind of breast carcinoma " selector " being made up of the DNA fragmentation of targeting breast cancer related gene Sudden change region.Isolated and purified patient with breast cancer's Circulating tumor DNA (ctDNA), the probe being designed to selector carries out solution hybridization capture, after high-flux sequence, finds the cancer gene variation that patient is special, and monitors tumor load.
First aspect, the invention provides a kind of specificity selector for human breast carcinoma detection, it is characterized in that comprising whole breast cancer related gene Sudden change region, it is made up of 496 example patient with breast cancer whole exon data in breast carcinoma sudden change driver gene and TCGA data base, a length of 0.365Mb.
Second aspect, the invention provides a kind of for the probe combinations containing above-mentioned specificity selector, it is characterized in that the customization of above-mentioned breast carcinoma selector is become probe, after isolated and purified patient circulates dissociative DNA, utilize this probe to carry out solution hybridization capture order-checking.
Concrete, a kind of probe combinations for the detection of human breast carcinoma Circulating tumor DNA of the present invention, it includes that breast carcinoma drives gene and the exon sequence of patient with breast cancer, and chromosome and initiation site thereof that described breast carcinoma driving gene and the exon sequence of patient with breast cancer are positioned at are the most as shown in the table:
Table 1
Further, the invention also discloses a kind of capture sequencing system for diagnosing human breast carcinoma, it includes that capture probe of the present invention, crossing system, sequencing device and circulation dissociative DNA extract test kit.
In the present invention, it is preferred to, when using above-mentioned capture sequencing system detection human breast carcinoma, comprise the following steps:
(1) use circulation dissociative DNA to extract test kit and extract plasma circulation dissociative DNA；
(2) the circulation dissociative DNA fragment ends reparation of extraction is added joint, build library；
(3) before capture, library is carried out PCR amplification purification；
(4) the sample library after amplification is hybridized with probe；
(5) remove uncombined sequence, then capture sequence is eluted；
(6) sample after capture is carried out again PCR amplification purification；
(7) high-flux sequence analysis.
Finally, the invention allows for described probe combinations and described capture sequencing system purposes in preparation diagnosis human breast carcinoma reagent.
Compared to prior art, beneficial effects of the present invention is embodied in:
High specificity: with the selector of the present invention and capture order-checking detection of platform patient with breast cancer's blood sample and healthy human blood's sample, only find sudden change after patient with breast cancer ctDNA sequencing analysis, display result is positive, and the sequencing analysis result of Healthy People circulation dissociative DNA is shown as negative.This explanation present invention has good specificity.
Highly sensitive: to use the method for the present invention to can detect that the 7-32ng DNA extracted from plasma of breast cancer patients sample, illustrate that the inventive method has the highest sensitivity.
High flux: the inventive method application high-flux sequence, the order-checking degree of depth of single sample can reach 10000x.
Non-invasive: it is peripheral blood in patients that the present invention invents required sample, it is not necessary to aspiration biopsy sample or cut sample this, it is convenient to collect, and repeatable high.
Detailed description of the invention
Further describe the present invention by the following examples, it should be understood that these embodiments are only used for the purpose of illustration, do not limit the scope of the invention.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, sees " molecular cloning " (Science Press, the second edition, 2002).
The design of the specificity selector that embodiment 1 one kinds detects for human breast carcinoma and the preparation of probe
1, the design of a kind of specificity selector for human breast carcinoma detection
The human breast carcinoma specificity selector of the present invention, contain 496 example patient with breast cancer whole exon data and breast carcinoma sudden change driver gene in TCGA (THE CANCER GENOME ATLAS) data base, a length of 0.365Mb, the concrete length of every part is as shown in table 2 below by breast carcinoma hypotype and gene distribution, is designed by bio information science and technology institute of Harbin Medical University and is verified.
Table 2
| Subtype | Selector(Mb) |
| Basal-like | 0.044069 |
| Her2 | 0.040817 |
| Luminal A | 0.216391 |
| Luminal B | 0.091488 |
| BRCA1 | 0.008220 |
| BRCA2 | 0.011386 |
| Total (after deduplication) | 0.365177 |
Concrete, 496 described example patient with breast cancer whole exon data and breast carcinoma sudden change driver gene include gene order as shown in table 3 below:
2, the preparation of probe
Sequence in above-mentioned table 3 is transferred to the preparation that Europe, Shanghai is readily accomplished probe.
Embodiment 2 one kinds is for diagnosing the capture sequencing system of human breast carcinoma
The capture order-checking platform of the present invention, the probe, crossing system, sequencing device (Roche Nimblegen) and the circulation dissociative DNA that prepare including embodiment 1 extract test kit (QIAamp Circulating Nucleic Acid Kit).
The application in diagnosis human breast carcinoma of the gene chip of embodiment 3 present invention
Capture order-checking platform used in the present invention is Roche Roche Nimblegen；It is QIAamp Circulating Nucleic Acid Kit that circulation dissociative DNA extracts test kit；Sequencing analysis is readily accomplished by Europe, Shanghai.
Biomaterial of the present invention is all from attached tumour hospital of Harbin Medical University Breast Surgery, and the tissue that all sample standard deviations have sufferers themselves to sign provides Informed Consent Form and ethics to prove.
Detection method:
One, plasma free Circulating tumor DNA extracts flow process (DNA extraction kit, QIAamp Circulating Nucleic Acid Kit)
1. collect blood samples of patients, 3000rpm with EDTA anticoagulant blood-collecting pipe, after centrifugal 10 minutes, separate supernatant, supernatant is centrifuged, 20000 × g again, after centrifugal 10 minutes, supernatant is separated standby.
2. draw in 100ul Proteinase K to 50ml centrifuge tube.
3. draw 1ml blood plasma to add in 50ml centrifuge tube.
4. draw 0.8ml Buffer ACL (containing 1.0ug carrier RNA) to after 50ml centrifuge tube, centrifugal pipe cap is buckled, pulse vortex 30 seconds.
Hatch 30 minutes for 5.60 degree
6. open centrifugal pipe cap, add 1.8ml Buffer ACB, more centrifugal pipe cap is buckled, the pulse vortex 15-30 second.
7. will be equipped with mixing the 50ml centrifuge tube ice bath 5 minutes of liquid.
8. QIAamp Mini column is placed on the Vacconnector of QIAvac 24Plus, QIAamp Mini column disposes the expansion pipe of a 20ml again.
9. the mixing liquid in 50ml centrifuge tube is added to in the QIAamp Mini column expanding pipe, utilize vacuum pump to be filtered by mixing liquid, then discard expansion pipe.
10. in QIAamp Mini column, add 600ul Buffer ACW1, utilize vacuum pump to be filtered by liquid.
11. add 750ul Buffer ACW2 in QIAamp Mini column, and liquid is filtered by this to utilize vacuum.
12. dehydrated alcohol adding 750ul in QIAamp Mini column, utilize vacuum pump to be filtered by liquid.
After the cap of QIAamp Mini column is buckled by 13., it is placed in a clean 2ml collecting pipe, high speed centrifugation (20000 × g) 3 minutes.
QIAamp Mini column is placed in another new collecting pipe by 14., opens cap, hatches 10 minutes for 56 degree.
QIAamp Mini column is placed in new 1.5ml centrifuge tube by 15., adds 30ul Buffer AVE on the film of QIAamp Mini column, after buckling cap, and incubated at room 3 minutes.
16. by 1.5ml centrifuge tube high speed centrifugation (20000 × g) 1 minute with QIAamp Mini column, eluting plasma free Circulating tumor DNA (ctDNA).
Two, capture order-checking flow process
Part I preparation of samples
The first step: DNA quality inspection
Note:ctDNA sample quality is up to standard: OD260/OD280=1.8-2.0, utilizes Qubit system to carry out DNA quantitatively.
A sequencing library built by each sample
Second step: AMPure XP beads purification of samples
1. by AMPure XP beads equilibrium at room temperature at least 30 minutes
70% ethanol of the most each preparation of samples 400 μ l, uses in step 8.
3. mixing bead suspension, observes reagent solid colour from color.
4. add 180 μ l uniform AMPure XP beads in each ctDNA sample, blow and beat 10 times and ensure to mix.
5. incubated at room sample 5 minutes.
6. sample cell or plate are placed on magnetic frame, stand to solution clarification (about 3-5 minute)
7. keep sample cell or plate on magnetic frame, be carefully removed supernatant, keep off when removing solution and contact magnetic bead.
8. continuing to be placed on magnetic frame by sample cell or plate, often pipe adds 200 μ l 70% ethanol.70% ethanol using new preparation is good to ensure experimental result.
9. waiting makes magnetic bead precipitate in 1 minute, then removes ethanol.
10. repeat 8,9 once.
11. seal with strip caps, the remaining ethanol of simple centrifugal collection, plate are put back to magnetic frame 30 seconds, remove remaining ethanol with P20.
Unsealed plate is put in PCR instrument drying sample by 12, and PCR instrument arranges 37 DEG C and keeps 3-5 minute, or until ethanol volatilizees totally completely.
13. add water that 50 μ l free nucleic acids pollute in each sample cell.
14. seal with strip caps, with vortex mixer mixing and are simply centrifuged to collect liquid.
15. room temperatures keep 2 minutes
Sample cell or plate are placed on magnetic frame holding 2-3 minute, until solution is clarified by 16..
17. transfers supernatant (general 48 μ l), in a new pipe, obtain ctDNA sample after purification, this time can abandon magnetic bead.If not carrying out next step, sealing leaves-20 DEG C in.
3rd step: end-filling
SureSelect XT Library Prep Kit ILM is utilized to carry out this step.
1. preparing suitable master mix according to table 4 below, experiment is carried out on ice, mixes with votex mixer, and sample is consistently placed on ice.
The preparation of table 4 end-filling reaction system
2. add 52 μ l master mix in the good ctDNA sample of purification, piping and druming mixing.
Sample is hatched according to following procedure, it is not necessary to heat lid on 3.PCR instrument.
After hatching 30min at 20 DEG C, maintain 4 DEG C.
4th step: AMPure XP beads purification of samples
1.AMPure XP beads balance at least 30 minutes to room temperature, the most frozen magnetic bead.
2. mixing magnetic bead and reagent are to color even.
3. add in 180 μ l uniform AMPure XP beads to 100 μ l end-filling DNA sample, blow and beat 10 mixing samples.
4. incubated at room 5 minutes.
5. sample cell or plate are put on magnetic frame, stand to solution clarification (about needing 3-5 minute).
6. sample cell or plate continue to be placed on magnetic frame, carefully remove and abandon supernatant, do not touch magnetic bead when removing supernatant.
7. continue plate or sample cell to be placed on magnetic frame, add 200 freshly prepared 70% ethanol of μ l.
8. wait 1 minute and precipitating to all magnetic beads, then remove ethanol.
9. repeat step 7-8 once.
10. with strip caps tube sealing, simple centrifugal collection remaining ethanol, sample cell or plate are put back to magnetic frame and stands 30 seconds, remove residue ethanol with P20 rifle head.
Plate or pipe are placed in PCR instrument and to be dried by 11, and PCR instrument arranges 37 degree and is incubated 3-5 minute, until ethanol volatilizees completely.
12. often add the water that 32 μ l free nucleic acids pollute in pipe.
13. mix with strip caps tube sealing, vortex mixer, and simply centrifugal collection liquid.
14. room temperatures are placed 2 minutes.
Sample cell or plate are placed on magnetic frame standing 2-3 minute by 15., until solution clarification.
16. transfer about 30 μ l supernatants in new pipe or PCR plate, obtain the ctDNA sample of end-filling after purification, and magnetic bead can abandon.
3 ' the ends of the 5th step: DNA add A
Carrying out this step experiment with SureSelect XT Library Prep Kit ILM, this step is all carried out on ice.
1. prepare the Adenylation master mix of suitable volumes according to table 5 below, experiment is carried out on ice, and vorgex mixer mixes.
The preparation of table 5 Adenylation master mix
The DNA sample (about 30 μ l) that the most often pipe end-filling purification are crossed adds 20ul Adenylation master mix.
3. piping and druming mixing.
4. PCR instrument is set according to following procedure, hatches sample.
After hatching 30min at 37 DEG C, maintain 4 DEG C.
6th step AMPure XP beads purification of samples
1. equilibrium at room temperature AMPure XP beads at least 30 minutes.
2. mixing magnetic bead is to solid colour.
3. AMPure XP beads to the 50 μ l adding 90 μ l mixings adds 3 ' end adds in the DNA sample of A, blows and beats 10 mixings.
4. incubated at room 5 minutes.
5. sample cell or plate are placed on magnetic frame and stand about 3-5 minute to solution clarification.
6. keep sample cell or plate on magnetic frame, carefully remove and abandon settled solution, keep off during operation and contact magnetic bead.
7. keeping sample cell or plate on magnetic frame, often pipe adds the ethanol that 200ul newly prepares.
8. stand 1 minute to precipitate to magnetic bead, remove ethanol.
9. repeat step 7-8 once.
10. with strip caps tube sealing, simple centrifugal collect residual ethanol, pipe or plate are put back to magnetic frame upper 30 second, removes residual ethanol.
Sample cell or plate are placed in PCR instrument by 11., and PCR instrument arranges 37 DEG C and is incubated 1-2 minute, or until residual ethanol is volatilized totally.
12. water that often pipe addition 15ul free nucleic acid pollutes.
13. seal with strip caps, vortex mixer mixing simply centrifugal collection liquid.
14. incubated at room 2 minutes.
Sample cell or plate are placed on magnetic frame standing 2-3 minute by 15., until solution clarification.
16. transfer 13 μ l settled solutions are in new PCR pipe or plate, and magnetic bead can abandon.
17. carry out next step experiment at once.
7th step: connect adaptor
SureSelect XT library Prep Kit ILM carries out the experiment of this step, and when carrying out this step, sample is put on ice
1. prepare Ligation master mix, the vortex mixer mixing of suitable volumes on ice according to table 6 below.
The preparation of table 6 Ligation master mix
2. add 37 μ l connection master mix to add in A purified DNA sample to each.
The most repeatedly blow and beat mixing.
4. in PCR instrument, hatch sample according to lower surface condition, not with heat lid.
After hatching 15min at 20 DEG C, maintain 4 DEG C.
If not carrying out next step experiment, after sample sealing, leave-20 DEG C in.
8th step: AMPure XP beads purification of samples
1. equilibrium at room temperature AMPure XP beads at least 30 minutes.
2. mixing magnetic bead and solvent are to color even.
The adaptor-ligated DNA sample of pipe 50 μ l the most often adds 90ul AMPure XP beads, piping and druming mixing.
4. incubated at room 5 minutes.
5. sample cell or plate are placed on magnetic frame, stand 3-5 minute and clarify to solution.
6. keep sample cell or plate on magnetic frame, be carefully removed and abandon settled solution, when removing solution, not touching magnetic bead.
7. keeping sample cell or plate on magnetic frame, often pipe adds 200 freshly prepared 70% ethanol of μ l.
8. stand 1 minute and wait beads precipitation, then remove ethanol.
9. repeat step 7-8 once.
10. seal with strip caps, simple centrifugal collection residual ethanol, sample cell or plate are put back to magnetic frame and stands 30 seconds, remove remaining ethanol.
11. sample cells or plate open-ended are placed in PCR plate, arrange temperature and are incubated 1-2 minute to 37 DEG C until residual ethanol is volatilized totally.
12. add the water that 32 μ l free nucleic acids pollute.
13. seal with strip caps, vortex mixer mixing simply centrifugal collection liquid.
14. room temperatures are placed 2 minutes.
Sample cell or plate are placed on magnetic frame 2-3 minute by 15., until solution clarification.
About 32 μ l supernatants are transferred in new pipe by 16., and magnetic bead can abandon.
9th step: amplification adaptor-ligated library
This step composition shows as follows, and all reagent melt on ice.
1. preparing the pre-capture PCR reaction mix of suitable volumes, carry out according to table 7 below on ice, vortex mixer mixes.
The preparation of table 7 pre-capture PCR reaction mix
The DNA sample that the most every 15 μ l purification are crossed adds the PCR Reaction mixture of 35 μ l, piping and druming mixing.
3. operation follow procedure:
Tenth step AMPure XP beads purification amplification library
1. equilibrium at room temperature AMPure XP beads at least 30 minutes.
2. mixing magnetic bead and reagent are to color even.
The most every 50 μ l amplification library samples add 90 AMPure XPbeads homogeneous for μ l, piping and druming mixing.
4. incubated at room 5 minutes.
5. sample cell or plate are placed on magnetic frame, stand 3-5 minute and clarify to solution.
6. keep sample cell or plate on magnetic frame, carefully remove and abandon supernatant, when removing solution, not touching magnetic bead.
7. keep sample cell or plate on magnetic frame, add 200 freshly prepared 70% ethanol of μ l.
8. stand 1 minute precipitation magnetic bead, then remove ethanol.
9. repeat step 7-8 once.
10. seal with strip caps, simple centrifugal collect residual ethanol, sample cell or plate are placed on magnetic frame upper 30 second, remove residue ethanol.
Sample cell or plate are placed in PCR instrument by 11., arrange temperature 37 DEG C insulation 1-2 minute or until residual ethanol is thoroughly volatilized totally.
12. often pipe add 30 μ l water.
13. sealings, vortex mixer mixing simply centrifugal collection liquid.
14. room temperatures are placed 2 minutes.
Sample cell or plate are placed on magnetic frame 2-3 minute by 15., until solution clarification.
16. transfers supernatant (about 30 μ l) are in new PCR pipe or plate, and magnetic bead abandons, and obtains DNA library after purification.If not going on subsequent experimental, sample is also put-20 DEG C of preservations by sealing.
Part II hybrid capture
First step DNA sample and the hybridization of probe
This step SureSelect Reagent Kit, melting of every kind of composition is carried out according to the requirement in form, and Vortex mixer mixes every kind of reagent, simple centrifugal collection liquid.Ingredient lists 8 is as follows:
Table 8 hybridizes reagent used
The 750ng sample that hybridization needs is dissolved in 3.4 μ l solution, initial concentration 221ng/ μ l.
1. pair off-the-shelf DNA library, if concentration is higher than 221ng/ μ l, is diluted to 221ng/ μ l concentration, needs 3.4 μ l altogether.
If 2. library concentration does not reaches 221ng/ μ l, being concentrated into 221ng/ μ l with vacuum drier, baking temperature must not be higher than 45 DEG C.
The library that 30 μ l build up is transferred to, in Ep pipe, stab several aperture, it is also possible to cover pipe with parafilm in lid by A, stabs several aperture.
Less than 45 DEG C concentration of DNA samples of B.
C adds water and adjusts to final concentration of 221ng/ μ l, notices that cleaning tube wall obtains the highest response rate.
D vortex mixer mixes and is centrifuged 1 minute.
3. transfer 3.4 μ l DNA sample library (750ng) are in new PCR pipe or plate, seal mouth and are placed on ice.
Caution: that must avoid follow-up 16-24 hour hatches the volatilization of middle sample
4. prepare hybridization buffer according to table 9 below under room temperature condition, if there being precipitation, within 5 minutes, can dissolve by 65 DEG C of incubations, hybridization buffer being placed on room temperature condition, until step 9 uses.
The preparation of table 9 hybridization buffer
*Prepare Hybridization Buffer for at least 5 reaction equivalents per run to allow accurate pipetting volumes.
5. preparing SureSelect Block Mix by mixing table 10 below composition, mixed liquor is placed on ice until step 6 uses.
The preparation of table 10 SureSelect Block Mix
6. the DNA library of couple each ready 3.4 μ l, adds the Sureselect Block Mix that 5.6 μ l volumes are prepared, piping and druming mixing according to upper table.
7. after sealing, the sample cell sealed or plate are placed in PCR instrument, the program of operation table 11 below:
Hot lid temperature: 105 DEG C, 65 DEG C of insulations, it is ensured that DNA+Block Mix sample is adding before other composition at least 65 DEG C of incubations more than 5 minutes.
Table 11 DNA+Block Mix thermocycling program before hybridization
8. size based on the capture library listed by table 12 below, prepares the SureSelect RNase Block of suitable volumes.The solution of the quantity needed for preparation hybridization, mixture to be placed on ice before step 9.
The preparation of table 12 RNase Block solution
Capture library mixture described by preparation process 9, it is ensured that 65 DEG C of incubations of 5 minutes.Mixture puts room temperature, until step 10 is mixed into sample.Will not be placed on room temperature containing the solution of SureSelect Capture Library.
9. mix according to table 13.Vortex is then centrifuged for collecting liquid for 5 seconds, until step 10 uses.
The preparation of table 13 Capture Library Hybridization Mix
10. keep DNA library+Block Mix to manage or plate is at 65 DEG C, the probe that 20 μ l Capture Library Hybridization Mix of addition step 9 preparation and 2 μ l embodiments 1 prepare, blow and beat 8-10 mixing.
Hybridization system is about 27-29 μ l now, depends on volatile quantity during 65 DEG C of incubations.
11. seal with Strip caps or plateLoc Thermal Microplate Sealer, it is ensured that the mouth of pipe completely encloses.Must fully seal to reduce evaporation, otherwise result reclaims impact.
Under the conditions of 12. 105 DEG C of heat lids, hatch hybridization mixture 16-24 hour for 65 DEG C.
Second step prepares Streptavidin and modifies magnetic bead
1.65 DEG C of preheating SureSelect Wash Buffer 2, for step 3.
2.vortex mixer acutely vibrates and mixes Dynabeads MyOne Streptavidin T1 magnetic bead, and magnetic bead can precipitate during depositing.
Magnetic bead the most first proceeds to new sample cell or plate, and each Hybridization samples needs to add the resuspended magnetic bead of 50ul.
4. washing magnetic bead:
A adds 200 μ l SureSelect Binding Buffer.
It is the most resuspended that B piping and druming makes bead make.
C will be equipped with the pipe of magnetic bead or plate is placed on magnetic frame.
D stands and clarifies to solution, removes and abandons supernatant.
E step A to D is repeated twice, altogether washing three times.
The 5.200 μ l Sureselect resuspended magnetic beads of Binding Buffer.
If Note has the magnetic capture system of large volume, Streptavidin MagneSphere can carry out large volume washing with Ep pipe, and after washing is good, 200 μ l portions carry out subpackage.
3rd step: Streptavidin MagneSphere capture hybrid dna
1. estimate and record incubation hybridization system liquor capacity after 24 hours.
2. when the solution drawn in hybridization system, hybrid pipe or plate should be maintained at 65 DEG C, often pipe should draw 25-29 μ l solution, and this solution and the 200 washed Streptavidin MagneSpheres of μ l is mixed.Slowly piping and druming makes magnetic bead the most resuspended.
3. sealing, sample cell or plate are placed on room temperature on Nutator mixer or the suitable instrument of function and keep 30 minutes, it is ensured that sample mix appropriateness.
4. simple Centrifuge A sample pipe or plate.
5. sample cell or plate are placed on magnetic frame, stand until solution clarification, remove and abandon supernatant.
The most resuspended 200 μ l SureSelect Wash Buffer 1, piping and druming is until magnetic bead is the most resuspended.
7. incubated at room sample 15 minutes.
The most centrifugal.
9. sample cell or plate are placed on magnetic frame, until solution clarification, remove and abandon supernatant.
Caution: whole washing process ensures bead re-suspension liquid temperature 65 DEG C, it is ensured that capture specificity.
Ensure that SureSelect Wash Buffer 2 is preheated to 65 DEG C before use.Experimentation can not use the instrument that temperature fluctuation is big.
10. wash magnetic bead with SureSelect Wash Buffer2.
The Wash Buffer 2 of 65 DEG C of preheatings of the resuspended 200 μ l of A, piping and druming is until magnetic bead is resuspended.
B seals, 65 DEG C of incubated samples pipes of PCR instrument or plate.
Sample cell or plate are placed on magnetic frame by C, stand and clarify to solution, remove and abandon supernatant.
D repeats step A to C 2 times, altogether washing 3 times.
Last washing terminates, it is ensured that all of wash buffer is removed.
11. add the water that 30 μ l free nucleic acids pollute, and piping and druming is with resuspended magnetic bead.
Keep all samples on ice until follow-up use.
In following amplification experiment, the DNA of capture remains attached on Streptavidin MagneSphere.
Part III: sample adds Index
First step 8-bp Indexes A01-H12 Indexing primers amplification capture library
This step agents useful for same is as listed by table 14 below: melt and vortex mix mixes and be placed on the most stand-by.
Table 14 is the reagent of PCR amplification after capture
*Do not use the PCR Reaction Buffer or dNTP mix from any other kit.
Each library expands with an index
1. specifying index, Indexes A01 to the H12 of each sample for DNA amplification library, the sample of a lane can not be with same indexing primer labelling.
2. prepare the PCR reactant mixture of suitable volumes on ice according to table 15 below
The preparation of the mixed liquor of PCR amplification after table 15 capture
The most each sample adds the PCR reactant mixture of 31 μ l
4. adding 5 μ l suitable Indexing Primer (SSEL 8bp Indexes A01-H12, backboard), each sample adds a primer in 16 or 96.
The most each PCR reaction adds DNA library sample.
A picks and places the PCR sample cell comprising 30ulbead-bound target-enriched DNA sample or plate (preceding step is ready) put on ice.
B blows and beats each DNA library until bead re-suspension liquid is homogeneous, then shifts 14 μ l samples in the PCR sample panel comprising PCRreaction mix and indexing primer or pipe.
C piping and druming mixing PCR reactant mixture.
-20 DEG C of the library that D residue is combined on magnetic bead is frozen standby.
6. carry out PCR amplification according to following procedure:
7.PCR EP (end of program), simple Centrifuge A sample pipe or plate.
Second step: purify with AMPure XP beads and capture the library expanded
1. equilibrium at room temperature AMPure XP beads at least 30 minutes, magnetic bead at any time can not be frozen.
2. preparing 400 freshly prepared 70% ethanol of μ l, step 9 uses.
The most resuspended AMPure XP beads is to color even.
The most every 50 μ l DNA amplification samples add 90 AMPure XPbead re-suspension liquid homogeneous for μ l.
5. piping and druming mixing.
Inspection guarantees that magnetic bead the most uniformly exists, the unification of each pipe color and the phenomenon not having magnetic bead to be layered.
6. incubation at room temperature 5 minutes.
7. sample panel or pipe being placed on magnetic frame, room temperature stands about 3-5 minute and clarifies to solution.
8. keep sample panel or pipe on magnetic frame, carefully remove and abandon supernatant, when removing supernatant, not touching magnetic bead.
9. keeping sample cell or plate on magnetic frame, often pipe adds 200 freshly prepared 70% ethanol of μ l.
10. stand 1 minute and wait magnetic bead precipitation, remove ethanol.
11. repeat step 9-10 once, it is ensured that finally remove all ethanol.
12. sample panel or channel closure, simple centrifugal collection residual ethanol, sample cell or plate are put back to magnetic frame 30 seconds, P20 rifle head removes residual ethanol.
Sample panel or pipe are placed in PCR instrument 37 DEG C of insulations 1-2 minute or until residual ethanol is volatilized completely by 13..
14. each pipes add 30 μ l free nucleic acid contaminant water.
15. sealings, vortex mixer mixing simply centrifugal collection liquid.
16. incubation at room temperature 2 minutes.
Sample panel or pipe are placed on magnetic frame upper 2 minute until solution is clarified by 17..
18. supernatants (about 30 μ l) are transferred to newly manage, and magnetic bead can abandon.
High-flux sequence is carried out after sample quality inspection is qualified.
Result:
Detect 5 example patients blood plasma to dissociate in Circulating tumor DNA to exist the catastrophe of PIK3CA, TP53, EGFR, AKT1 and PTEN, screening out the SNP that peripheral blood cells detects, the average mutation frequency that 5 kinds of genes of each patient detect in blood plasma see table 16:
Table 16
| Patient | Average mutation frequency (%) | Hypotype |
| 1 | 4.24 | Luminal B Her2 |
| 2 | 17.69 | Basal-like |
| 3 | 12.75 | Her2 |
| 4 | 8.04 | Her2 |
| 5 | 35 | Her2 |
Table 17 below is the initial data (a part of) of detection:
Table 17
The ctDNA concentration that blood plasma detects see table 18:
Table 18
With the selector of the present invention and capture order-checking detection of platform patient with breast cancer's blood sample and healthy human blood's sample, only find sudden change after patient with breast cancer ctDNA sequencing analysis, display result is positive, and the sequencing analysis result of Healthy People circulation dissociative DNA is shown as negative.This explanation present invention has good specificity.The method of the present invention can detect that, the 7-32ng DNA extracted from plasma of breast cancer patients sample, illustrates have the highest sensitivity.This method application high-flux sequence, the order-checking degree of depth of single sample can reach 10000x.
The foregoing is only the preferred embodiments of the present invention, be merely illustrative for the purpose of the present invention, and nonrestrictive；Those of ordinary skill in the art understand, it can be carried out many changes, amendment, even equivalence change, but fall within protection scope of the present invention in the spirit and scope that the claims in the present invention are limited.
Claims (5)
1. for the probe combinations of human breast carcinoma Circulating tumor DNA detection, it is characterised in that include that breast carcinoma is driven
Dynamic gene and the exon sequence of patient with breast cancer, described breast carcinoma drives gene and the outer of patient with breast cancer to show
Chromosome and initiation site thereof that subsequence is positioned at are the most as shown in the table:
2. the capture sequencing system being used for diagnosing human breast carcinoma, it is characterised in that include described in claim 1
Probe combinations, crossing system, sequencing device and circulation dissociative DNA extract test kit.
Capture sequencing system the most as claimed in claim 2, it is characterised in that when human breast carcinoma detects, bag
Include following steps:
(1) use circulation dissociative DNA to extract test kit and extract plasma circulation dissociative DNA；
(2) the circulation dissociative DNA fragment ends reparation of extraction is added joint, build library；
(3) before capture, library is carried out PCR amplification purification；
(4) the sample library after amplification is hybridized with probe；
(5) remove uncombined sequence, then capture sequence is eluted；
(6) sample after capture is carried out again PCR amplification purification；
(7) high-flux sequence analysis.
4. the purposes in preparation diagnosis human breast carcinoma reagent of the probe combinations described in claim 1.
5. the purposes in preparation diagnosis human breast carcinoma reagent of the capture sequencing system described in claim 2.
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| CN117344014A (en) * | 2023-07-19 | 2024-01-05 | 上海交通大学医学院附属瑞金医院 | Pancreatic cancer early diagnosis kit, method and device thereof |
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