CN107075732A - The construction method of dissociative DNA sequencing library - Google Patents

The construction method of dissociative DNA sequencing library Download PDF

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CN107075732A
CN107075732A CN201480082482.8A CN201480082482A CN107075732A CN 107075732 A CN107075732 A CN 107075732A CN 201480082482 A CN201480082482 A CN 201480082482A CN 107075732 A CN107075732 A CN 107075732A
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dissociative dna
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赵鑫
崔路漫
刘耿
宋彬
侯勇
吴逵
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MGI Tech Co Ltd
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Abstract

The invention discloses a kind of construction method of dissociative DNA sequencing library.This method includes：1) end reparation is carried out to dissociative DNA, obtains the dissociative DNA being repaired to end, the concentration of T4DNA polymerases is that the concentration of 1.5 3U/50 μ L, T4 polynueleotide kinases is 5 10U/50 μ L in the system；2) end plus A are carried out to the dissociative DNA that end is repaired, the concentration for obtaining Klenow fragments (3 ' 5 ' exo) in end plus A dissociative DNA, the system is 2.5 5U/50 μ L；3) A dissociative DNA adjunction head is added to obtain the dissociative DNA of belt lacing to end, the concentration of the system Chinese library joint is that the concentration of 20 40pmol/50 μ L, T4DNA ligases is 1200 2400U/50 μ L；4) dissociative DNA of belt lacing is expanded, dissociative DNA library is obtained.

Description

The construction method of dissociative DNA sequencing library Technical field
The present invention relates to the construction methods of dissociative DNA sequencing library.
Background technique
In blood of human body, dissociative DNA (cell-free DNA containing histocyte release, cfDNA), Tumour DNA (circulating tumor DNA wherein comprising tumor tissues release, ctDNA), cancer patient's blood plasma dissociative DNA contains various mutations gene relevant to tumour.Clinically, the ctDNA of cancer patient is mainly derived from tumor cell secretion, apoptosis or necrosis, and it is 20 times of normal person or more that concentration is average up to 180ng/mL.Studies have shown that the average length of ctDNA is 140-170bp, gene mutation situation can directly reflect the gene mutation situation [1,2] of tumor tissues.With the development of second generation sequencing technologies, the sequencing technologies based on genome mutation detection peripheral blood sample have been applied to the clinical level such as pre-natal diagnosis；And in haematological malignancies, the Somatic stricrure variation of height recurrence has been found to be effective molecular diagnostic markers object, including BCR-ABL proto-oncogene, immunoglobulin gene, T cell receptor gene, RAR α gene etc., analysis method based on real-time quantitative PCR can be reset with the target gene of free cancer cell in quantitative detection peripheral blood, these molecular diagnostic markers objects can be used for monitoring in real time tumor development situation, Neoplasm residual amount and long-term clinical management etc. after detection operation.Nitzan Rosenfeld team demonstrates the technology using new technology-target fragment deep sequencing method in 62 parts of plasma dna samples of 37 ovarian cancer patients effectively to monitor tumour by detection ctDNA gene mutation, its result is consistent with digital PCR testing result height, even more it is better than digital PCR in terms of finding unknown mutation, and the technology is easy to operate compared with digital round pcr.Then, Nitzan Rosenfeld team chooses 30 patients to make a variation with PIK3CA or TP53 point mutation or gene structure from 52 metastatic breast cancer patients, to 141 parts of plasma dna sample gene mutation situations and other two detection biomarker, that is CA15-3 level and CTC quantity compares, detect plasma dna mutation sensitivity and and disease progression situation the goodness of fit all compared with other two biomarker high [3,4].2013, Murtaza et al. tracing detection 2 breast cancer, the variation situation of 3 oophoromas and 1 the lung cancer dissociative DNA in blood plasma, and elaborate the change of the catastrophe in genes such as EGFR, RB1 and MED1 after different times drug therapy, prompt the genetic mutation after treating in monitoring cancer peripheral blood in patients blood plasma that doctor can be instructed to find the case where recurring and shifting in time and adjust therapeutic scheme [5] in time.
Tumor incidence and the death rate are in rising trend always in recent years, and oncotherapy is world medicine significant challenge.Many tumour conventional chemotherapy effects are poor, and prognosis mala is the clinical important problem of puzzlement, and drug resistance of tumor is then the key factor of chemotherapy of tumors failure, not only generates serious toxic side effect, delay treatment, and brings financial burden to patient.Such as, EGFR, which is mutated positive tumor, has preferable sensibility and poor to other chemotherapy drug susceptibilities to Tarceva or Gefitinib, needs to detect patient's EGFR catastrophe before the chemotherapeutics medication such as Iressa and Erlotinib.Therefore, the detection of drug susceptibility related gene is carried out to tumor patient, optimal chemotherapeutics and therapeutic scheme is selected for Different Individual, oncotherapy effect, realization Personalized medicine are significant to improving.And clinically obtain specimens frequently with the methods of tissue needle biopsy carries out genetic test again, not only causes severe trauma to patient, but also cumbersome, can not achieve the purpose that drug susceptibility real-time monitoring.
Conventional ILLUMINA Hiseq2000 builds library and needs a large amount of gDNA (1-3 microgram), and it is micro- not to be suitable for blood plasma Amount fragmentation gDNA (about a few nanograms) builds library.
Bibliography
1.Diehl,F.and M.Li,et al.(2005)."Detection and quantification of mutations in the plasma of patients with colorectal tumors."PNAS 102:16368-16373.
2.Diehl,F.and K.Schmidt,et al.(2008)."Circulating mutant DNA to assess tumor dynamics."Nat Med 14(9):985-90.
3.Forshew,T.and M.Murtaza,et al.(2012)."Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA."SciTransl Med 4(136):136ra68.
4.Dawson,S.J.and D.W.Tsui,et al.(2013)."Analysis of circulating tumor DNA to monitor metastatic breast cancer."N Engl J Med 368(13):1199-209.
5.Murtaza,M.and S.J.Dawson,et al.(2013)."Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA."Nature 497(7447):108-12.
Disclosure of the invention
The present invention provides the construction methods of dissociative DNA sequencing library.
The construction method of dissociative DNA sequencing library provided by the present invention, comprising:
1) end reparation is carried out to dissociative DNA, obtains the dissociative DNA being repaired to end；It repairs and is carried out in the end reparation system containing archaeal dna polymerase and nucleoside monophosphate kinase in the end, concentration of the archaeal dna polymerase in the end reparation system can be 1.5U-3U/50 μ L, concretely 1.5U/50 μ L, concentration of the nucleoside monophosphate kinase in the end reparation system can be 5U-10U/50 μ L, concretely 5U/50 μ L；
2) end is carried out to the dissociative DNA that the end is repaired and add " A ", obtain the dissociative DNA that end adds " A "；The end adds " A " to add in " A " system and carry out in the end containing Klenow segment (3 ' -5 ' exo-) (Klenow (3 ' -5 ' exo-)), the Klenow segment (3 ' -5 ' exo-) adds the concentration in " A " system that can be 2.5U-5U/50 μ L in the end, concretely 5U/50 μ L；
3) the dissociative DNA linking library connector of " A " is added to obtain the dissociative DNA of belt lacing to the end；The linking library connector carries out in the adjunction head system containing library connector and DNA ligase, and concentration of the library connector in the adjunction head system is 20-40pmol/50 μ L, concretely 20pmol/50 μ L；Concentration of the DNA ligase in the adjunction head system can be 1200U-2400U/50 μ L, concretely 1200U/50 μ L；
4) dissociative DNA for expanding the belt lacing, obtains dissociative DNA library；The recurring number of the amplification can be 8-12, concretely 10.
In the construction method of above-mentioned dissociative DNA sequencing library, the volume that system is repaired in the end can be 20-100 μ L, concretely 50 μ L.
In the construction method of above-mentioned dissociative DNA sequencing library, the archaeal dna polymerase is T4 archaeal dna polymerase, and the nucleoside monophosphate kinase is T4 polynueleotide kinase, and the DNA ligase is T4 DNA ligase.
In the construction method of above-mentioned dissociative DNA sequencing library, contain Klenow segment (Klenow Fragment) in the end reparation system, the Klenow segment can be 2.5-5U/50 μ L in the concentration that system is repaired in the end, concretely 2.5U/50 μ L.
In the construction method of above-mentioned dissociative DNA sequencing library, the end is repaired can be in 20 DEG C of progress 30min.
In the construction method of above-mentioned dissociative DNA sequencing library, the end adds " A " can be in 37 DEG C of progress 30min.
In the construction method of above-mentioned dissociative DNA sequencing library, the linking library connector can be in 20 DEG C of progress 15min.
In the construction method of above-mentioned dissociative DNA sequencing library, 4) amplification carries out in the amplification system containing Platinum Pfx archaeal dna polymerase (Platinum Pfx DNA polymerase), concentration of the Platinum Pfx archaeal dna polymerase in the amplification system is 2U-4U/50 μ L, concretely 2U/50 μ L.
In the construction method of above-mentioned dissociative DNA sequencing library, the circulating temperature program of the amplification is first 94 DEG C of 20s, then 62 DEG C of 40s, last 72 DEG C of 50s.
In the construction method of above-mentioned dissociative DNA sequencing library, the construction method may include the step of extracting the dissociative DNA from body fluid.
Application of the construction method of above-mentioned dissociative DNA sequencing library in the sequencing of dissociative DNA also belongs to protection scope of the present invention.
Application of the construction method of above-mentioned dissociative DNA sequencing library in detection dissociative DNA variation situation also belongs to protection scope of the present invention.
Above, the dissociative DNA can be the dissociative DNA (in vitro body fluid dissociative DNA) in body fluid, such as dissociative DNA in blood (in vitro dissociative DNA in blood), plasma DNA (in vitro plasma DNA).
Experiments have shown that, the sequencing analysis method of library construction is carried out using the construction method of dissociative DNA sequencing library of the invention, the target area capture coverage of human peripheral blood plasma DNA averagely reaches 98.7%, target area average capture efficiency has reached 61.2%, compared with the similar capture rate for studying average 10-15% or so, the capture rate of the sequencing analysis method is even up to the capture rate that cell line/tissue samples etc. have the capture of the target area of complete genome group DNA and the preferable sample of quality to build library.Sequencing analysis method of the invention can detect the point mutation of patients with lung cancer EGFR, short insertion/deletion, the copy numerical mutation of MET and EGFR.Sequencing analysis method of the invention can detect the frequency proportions for the mutation that frequency is extremely low in peripheral blood from patients with lung cancer blood plasma and the frequency of mutation ratio of Drug-resistant and sensitivity.
Detailed description of the invention
Fig. 1 is that peak figure is detected in peripheral blood from patients with lung cancer plasma DNA library 2100.
Fig. 2 is that peripheral blood from patients with lung cancer plasma DNA target area captures coverage distribution.
Fig. 3 is the distribution of peripheral blood from patients with lung cancer plasma DNA target area capture rate.
Fig. 4 is the change situation of clinical application MET gene copy number MET gene copy number in same patients with lung cancer difference disease progression: 1 represents I phase of disease progression, and 2 represent II phase of disease progression, and 3 represent III phase of disease progression.
The best mode to carry out an invention
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experimental method in following embodiments is unless otherwise specified conventional method.Test material as used in the following examples, It unless otherwise specified, is to be commercially available from routine biochemistry reagent shop.Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
The construction method for the dissociative DNA sequencing library being provided below is the method for the suitable building dissociative DNA sequencing library that the following steps in the construction method to the DNA sequencing library of ILLUMINA Hiseq2000 improve:
1) in the operation for carrying out end reparation to DNA, the concentration of T4 archaeal dna polymerase in the reaction system is respectively 1.5U-3U/50 μ L, concretely 1.5U/50 μ L；The concentration of T4 polynueleotide kinase in the reaction system can be 5U-10U/50 μ L, concretely 5U/50 μ L；
2) DNA progress end adds in the operation of " A ", and the concentration of Klenow segment (3 ' -5 ' exo-) in the reaction system can be 2.5U-5U/50 μ L, concretely 5U/50 μ L；
3) DNA is attached in the operation of library connector, concentration of the library connector in adjunction head system is 20-40pmol/50 μ L, concretely 20pmol/50 μ L；Concentration of the T4 DNA ligase in adjunction head system can be 1200U-2400U/50 μ L, concretely 1200U/50 μ L；
4) in the operation of amplified band linker DNA, the recurring number of amplification increases to 8-12 circulation, and concretely 10.
The construction method of the dissociative DNA sequencing library of the application is illustrated by taking patients with lung cancer as an example below.
10 × Polynucleotide Kinase (PNK) Buffer, dNTP Solution Set, T4 DNA Polymerase, Klenow Fragment, T4Polynucleotide Kinase, Klenow (3 ' -5 ' exo-) kit, dATP, 2 × Rapid l igation buffer, T4 DNA Ligase (Rapid) are the product of Enzymatics company；PE Adapter oligo mix,Invitrogen Dynabeads M-280,Pfx DNA Polymerase kit and Cot-1DNA are the product of Invitrogen company；SnoMagTMCirculating DNA kit is the product of SNOVA Biotechnologies company；2 × SC Hybridization Buffer and SC Hybridization Component A is the product of Roche Nimblegen company；SeqCap EZ Hybridization and Wash kit is the product of ROCHE DIAGNOSTICS company；AmpureXP beads is the product of AGENCOURT company；Flowcell primers F and Flowcell primers R is Illumina Products.
Embodiment 1, the construction method of peripheral blood from patients with lung cancer plasma DNA sequencing library and its application
The sequencing analysis method for present embodiments providing peripheral blood plasma DNA, including constructing the sequencing of peripheral blood from patients with lung cancer plasma DNA sequencing library and peripheral blood from patients with lung cancer plasma DNA sequencing library and analyzing the two steps.
Library constructing method is the construction method of the suitable peripheral blood from patients with lung cancer plasma DNA sequencing library improved on the basis of Hiseq library constructing method.
One, peripheral blood from patients with lung cancer plasma DNA sequencing library is constructed
Building peripheral blood from patients with lung cancer plasma DNA sequencing library includes the following steps (one), (two), (three), (four) and (five).
(1) extraction of peripheral blood from patients with lung cancer plasma DNA
Using SnoMagTMCirculating DNA kit carries out the extraction of peripheral blood from patients with lung cancer plasma DNA, specific as follows:
1, it takes 200 μ L peripheral blood from patients with lung cancer blood plasma to manage in 1.5mLEP, 600 μ L buffer LSB is added (LSB buffer).
2,20 μ LNanoMag Circulating Beads are added to mix, are placed at room temperature for 10min, every 2-3min mixes primary.
3, EP pipe is placed on magnetic frame and adsorbs 1min, abandon supernatant.
4, it removes EP pipe and 150 μ L Buffer WA (WA buffer) is added, mix.
5, EP pipe is placed on magnetic frame and adsorbs 1min, abandon supernatant.
6, it removes EP pipe and 150 μ L, 75% ethyl alcohol is added, mix.
7, EP pipe is placed on magnetic frame and adsorbs 1min, abandon supernatant.
8, it is primary to repeat step 6-7.
9, the magnetic bead 5min in drying at room temperature EP pipe.
10,32 μ L elution buffer (eluent) are added and mix magnetic bead, be stored at room temperature 5min.
11, EP pipe is placed on magnetic frame and adsorbs 1min, obtained supernatant is peripheral blood from patients with lung cancer plasma DNA solution, shifts the supernatant to new 1.5mL EP pipe.Measure the concentration of peripheral blood from patients with lung cancer plasma DNA in 16 peripheral blood from patients with lung cancer plasma DNA solution.The results are shown in Table 1:
The concentration of table 1, peripheral blood from patients with lung cancer plasma DNA
(2) end reparation is carried out to peripheral blood from patients with lung cancer plasma DNA, obtains the dissociative DNA being repaired to end
System is repaired in 50 ends μ L that end restorative procedure is prepared according to table 2, and in the 50 μ L end reparation system, it be the concentration of 5U/50 μ L, Klenow segment is 2.5U/50 μ L that the concentration of T4 archaeal dna polymerase, which is the concentration of 1.5U/50 μ L, T4 polynueleotide kinase,.
50 ends the μ L reparation system of table 2, end restorative procedure
50 end μ L dummies are tied up into 20 DEG C of incubation 30min in Thermomixer constant temperature dry bath device, obtain the dissociative DNA solution being repaired to end.The dissociative DNA being repaired using the end that 90 μ L AmpureXP beads purifying magnetic beads for purifying obtains, and elute the dissociative DNA that the end is repaired from AmpureXP beads with 36 μ L Elution buffer (elution buffer) and obtain the dissociative DNA solution that end after purification is repaired.
(3) end is carried out to the dissociative DNA that end is repaired and adds " A ", obtain the dissociative DNA that end adds A
End adds " A " method to add " A " system according to 50 ends μ L that table 3 is prepared, which adds in " A " system, and the concentration of Klenow segment (3 ' -5 ' exo-) is 5U/50 μ L.
Table 3, end add 50 ends μ L of " A " method to add " A " system
The dissociative DNA solution that the end of step (2) is repaired 34μL
10 × blue buffer (10 × blue buffer) 5μL
dATP(1mM) 10μL
Klenow(3’-5’exo-)(5U/μL) 1μL
Total volume 50μL
50 ends μ L of end plus " A " method are added into " A " system 37 DEG C of incubation 30min in Thermomixer constant temperature dry bath device, obtain the dissociative DNA solution that end adds " A ".The dissociative DNA of " A " is added using the end that 90 μ L AmpureXP beads purifying obtains, and elutes the end from AmpureXP beads with 24 μ L Elution buffer and the dissociative DNA of " A " is added to obtain the dissociative DNA solution that end adds " A ".
(4) add the both ends of the dissociative DNA of " A " to connect the library Illumina Hiseq2000 connector to end, obtain the dissociative DNA of belt lacing
In the reaction system, it is 1200U/50 μ L that the concentration of library connector (PE Adapter oligo), which is the concentration of 20pmol/50 μ L, T4 DNA ligase,.
50 μ L adjunction head systems of table 4, both ends linking library jointing method
The end of the purifying of step (3) adds the dissociative DNA solution of " A " 22.5μL
2 × Rapid ligation buffer (2 × efficiently connect buffer) 25μL
PE Adapter oligo mix (double end fitting mixed liquors) (40 μM) 0.5μL
T4 DNA Ligase (Rapid) (T4 DNA ligase (efficient)) (600U/ μ L) 2μL
Total volume 50μL
By 50 μ L adjunction head systems of both ends linking library jointing method in Thermomixer constant temperature dry bath device 20 DEG C of incubation 15min, obtain the dissociative DNA solution of belt lacing.Using the dissociative DNA for the belt lacing that 75 μ L AmpureXP beads purifying obtains, and the dissociative DNA solution of belt lacing after purification is obtained from the dissociative DNA that AmpureXP beads elutes the belt lacing with 34 μ L Elution buffer.
(5) PCR pre- amplified reaction in library is carried out to the dissociative DNA of belt lacing, constructs peripheral blood from patients with lung cancer plasma DNA library
PCR pre- amplification method in library prepares the 50 pre- amplification reaction systems of μ L library PCR according to table 5, and the concentration of Platinum Pfx archaeal dna polymerase (Platinum Pfx DNA polymerase) is 2U/50 μ L in the pre- amplification reaction system of 50 μ L library PCR.
The 50 pre- amplification reaction systems of μ L library PCR of table 5, the pre- amplification method of library PCR
The dissociative DNA solution of the belt lacing of the purifying of step (4) 32.2μL
10 × PfxAmplification Buffer (10 × Pfx amplification buffer) 5μL
Public primer (10 μM) (consensus primer) 4μL
Indexprimer (10 μM) (Tag primer) 4μL
MgSO4 (concentration 50mM) (magnesium sulfate) 2μL
DNTP Solution Set (every kind of dNTP concentration is 10mM) (dNTP mixed liquor) 2μL
Platinum Pfx DNA polymerase (2.5U/ μ L) (Pfx archaeal dna polymerase) 0.8μL
Total volume 50μL
By the 50 pre- amplification reaction systems of μ L library PCR of the pre- amplification method of library PCR in advance in PCR instrument in 94 DEG C of preheating 2min, it is denaturalized template DNA sufficiently subsequently into the amplification cycles stage；The circulating temperature program of amplification is that 94 DEG C of holding 20s make template denaturation, and then 62 DEG C of holding 40s make primer and template annealing, and last 72 DEG C of holdings 50s extends primer in template, synthetic DNA, a circulation is completed, is recycled as progress 10 times altogether, accumulates the DNA fragmentation of amplification largely；Subsequent 72 DEG C of holdings 10min it is complete to guarantee that product extends, 4 DEG C of preservations.The peripheral blood from patients with lung cancer plasma DNA library for expanding preparation in advance by PCR is obtained into peripheral blood from patients with lung cancer plasma DNA library using 60 μ L AmpureXP beads purifying, and it is obtained peripheral blood from patients with lung cancer plasma DNA library (abbreviation Pre-PCR library) with 22 μ L Elution buffer (elution buffer) from the peripheral blood from patients with lung cancer plasma DNA of AmpureXP beads (purifying magnetic bead) elution amplification.The concentration of DNA in the library is quantitatively determined using QubitdsDNA HS assay kit (the high quick detection kit of Qubit double-stranded DNA) to peripheral blood from patients with lung cancer plasma DNA library.
The result shows that the concentration in 16 libraries Pre-PCR of peripheral blood from patients with lung cancer plasma DNA is as shown in table 6:
Table 6: the Pre-PCR library concentration of plasma DNA
Sample ID number Concentration (ng/ μ L) Sample ID number Concentration (ng/ μ L)
1 6.86 9 15.5
2 13.35 10 19.96
3 16.16 11 17.87
4 17.7 12 24.51
5 8.47 13 13.73
6 8.65 14 6.8
7 22.09 15 17.36
8 18.55 16 20.27
Two, the sequencing and analysis of peripheral blood from patients with lung cancer plasma DNA sequencing library
(1) peripheral blood from patients with lung cancer plasma DNA library and Nimblegen chip hybridization carry out the capture of target area domain dna
1, it by the Pre-PCR l ibrary of (five) in step 1, carries out being mixed to get a mixing pit progress Library hybridization in such a way that the Pre-PCR library of 16 patients with lung cancer is mixed, finally obtains 1 upper machine library.The content of DNA is 1000ng in one mixing pit, and the Pre-PCR library content of every patients with lung cancer is 62.5ng.
2, according to Hybrid Library system is prepared shown in table 7, which is obtained into dry Hybrid Library product in 60 DEG C of concentration 15min using vacuum concentration instrument.
Table 7, peripheral blood from patients with lung cancer plasma DNA and Nimblegen chip hybridization library system
Pre-PCR library Total 1000ng
People Cot-1 DNA (1mg/mL) 5μL
The public Block of P1 (200 μM) (public closing primer) 4μL
Indexblock (200 μM) (label closing primer) 4μL
3, hybridization buffer reaction system is configured according to table 8.
Table 8, hybridization buffer reaction system
2 × SC Hybridization Buffer (2 × SC hybridization buffer) 7.5μL
SC Hybridization Component A (SC hybridizes component A) 3μL
Total volume 10.5μL
4, the hybridization buffer reaction system that the 10.5 prepared steps 3 of μ L are added in dry Hybrid Library product is obtained to step 2, in 95 DEG C of processing 10min, obtains the Hybrid Library system solution being dissolved in hybridization buffer reaction system.
5, library and probe hybridization reaction: taking 4.5 μ L Nimblegen chips that step 4 Hybrid Library system solution is added, and in 47 DEG C of hybridization 72h, obtains hybrid product solution.
(2) elution of peripheral blood from patients with lung cancer plasma DNA target area domain dna is carried out after hybridizing
1, Invitrogen Dynabeads M-280 magnetic bead is acutely shaken to mixing to be resuspended, it is spare.
2,100 μ L Invitrogen Dynabeads M-280 magnetic beads of addition wash magnetic bead, steps are as follows into 1.5mL EP pipe:
1) 200 μ L Binding Buffer (Binding buffer) are added, acutely Invitrogen Dynabeads M-280 is resuspended in concussion 5s, it is placed on magnetic frame and stands 2min, it is carefully inhaled after liquid is clarified completely and abandons supernatant, obtain Invitrogen Dynabeads M-280 precipitating；
2) it repeats step 1) 1 time, washs 2 times altogether；
3) 100 μ L Beads Wash Buffer (magnetic bead washing buffer) are added into the EP pipe of the 1.5mL precipitated equipped with Invitrogen Dynabeads M-280 and the magnetic bead is resuspended.
3, the magnetic bead after resuspension is gone in the sample tube of the hybrid product solution containing step (1), is placed in 47 DEG C of incubation 45min in PCR instrument, mixed once every 15min, altogether three times.
4, the magnetic bead suspension after incubation is transferred in new 1.5mLEP pipe, is placed on magnetic frame after standing and abandons supernatant.
5, Wash Buffer I (washing buffer I) (47 DEG C) 100 μ L is added into the magnetic bead of step 4 precipitating, mixes sample, is placed on magnetic frame and stands 3-5min, clarified completely to liquid, careful inhale abandons supernatant.
6, Stringent buffer (Stringent buffer) (47 DEG C) 200 μ L is added into the magnetic bead of step 5 precipitating, it mixes, it is placed in 47 DEG C of incubation 5min in Thermomixer, brief centrifugation, it is placed on magnetic frame and is placed in 3-5min, it is clarified completely to liquid, careful inhale abandons supernatant.
7, step 6 is repeated to operate 1 time, totally 2 times.
8, Wash Buffer I (RT) 200 μ L is added into the magnetic bead of step 7 precipitating, is placed in Thermomixer and is centrifuged 2min in 20 DEG C of 1400rpm, be placed on magnetic frame and stand 3-5min, clarified completely to liquid, careful inhale abandons supernatant.
9, step 8 is repeated to operate 2 times, totally 3 times.
10,140 μ L Nuclease Free-water (seedless sour water) are added into the magnetic bead of step 9 precipitating and magnetic bead is resuspended, obtain the magnetic bead suspension containing capture target area domain dna.
(3) bridge-type PCR amplification is carried out to the target area domain dna of capture
1,100 μ L bridge-type pcr amplification reaction systems are configured in 1.5mL EP pipe according to table 9.
Table 9, bridge-type pcr amplification reaction system
The magnetic bead suspension containing capture target area domain dna of step (2) 70μL
10 × PfxAmplification Buffer (10 × Pfx amplification buffer) 10μL
Flowcellprimers F (10 μM) (Flowcell primer is positive) 5μL
Flowcellprimers R (10 μM) (Flowcell primer is reversed) 5μL
MgSO4(50mM) (magnesium sulfate) 4μL
DNTP Solution Set (10mM) (dNTP mixed liquor) 4μL
Platinum Pfx DNA polymerase (2.5U) (Pfx archaeal dna polymerase) 2μL
Total volume 100μL
By 100 μ L bridge-type pcr amplification reaction systems in advance in PCR instrument in 94 DEG C of preheating 2min, it is denaturalized template DNA sufficiently subsequently into the amplification cycles stage；The circulating temperature program of amplification is that 94 DEG C of holding 15s make template denaturation, and then 58 DEG C of holding 30s make primer and template annealing, and last 72 DEG C of holdings 30s extends primer in template, synthetic DNA, a circulation is completed, is recycled as progress 15 times altogether, accumulates the DNA fragmentation of amplification largely；Subsequent 72 DEG C of holdings 5min, it is complete to guarantee that product extends, and 4 DEG C of preservations obtain bridge-type pcr amplification product.
2, bridge-type pcr amplification product purifies: the PCR pipe of the bridge-type pcr amplification product equipped with step 1 is placed on magnetic frame, 3-5min is stood to clarification, careful supernatant of drawing is transferred in new 1.5mLEP pipe, 120 μ L AmpureXP beads purifying supernatant is added, finally with 31 μ L Elution buffer (eluent) eluted products, obtain finally going up machine library.
3, upper machine library quality inspection: Library Quality detection is carried out using Agilent 2100Bioanalyzer and quantitative fluorescent PCR (QPCR), 2100 detection peak figures are as shown in Figure 1, testing result is as shown in table 10:
Table 10: library 2100 and QPCR testing result
QPCR result is less than 500nM greater than 5nM, and mass concentration is greater than 0.5ng/ μ L, and segment ranges 250-330bp meets above confidential ask.Should the result shows that, most of segment in library concentrates on 280-300bp, and concentration meets machine sequencing and requires.
(4) upper machine sequencing is carried out to the target area domain dna of capture and data is analyzed
1, machine is sequenced on Illumina Hiseq2000: being subject to QPCR detectable concentration, program is sequenced according to machine Illumina Hiseq2000 on and carries out upper machine sequencing to the bridge-type PCR product of the purifying in step (3).
2, lower machine data analysis: data filtering, comparing and variation are carried out to upper machine sequencing data and tested and analyzed.
2.1 target areas capture coverage and capture rate
The target area capture coverage distribution of sequencing analysis method is as shown in Fig. 2, from figure 2 it can be seen that target area capture coverage averagely reaches 98.7%.Illustrate that the data that this method obtains almost have covered all target areas, the needs of subsequent abrupt climatic change can be fully met.The target area capture rate distribution of the sequencing analysis method is as shown in Figure 3, average capture efficiency has reached 61.2%, compared with the similar capture rate for studying average 10-15% or so, the capture rate of the sequencing analysis method is even up to the capture rate that cell line/tissue samples etc. have the capture of the target area of complete genome group DNA and the preferable sample of quality to build library.
2.2 abrupt climatic change results
Clinically, the copy number detection of the point mutation of patients with lung cancer EGFR, short insertion/deletion, MET and EGFR have become using the inspection that must be carried out before targeted drug Iressa and Erlotinib treatment.The above-mentioned catastrophe that these genes have been also detected that in the peripheral blood from patients with lung cancer blood plasma of sequencing analysis is carried out using the sequencing analysis method, simultaneously by the result compared with patient clinical information carries out, as a result, it has been found that the abrupt climatic change result of the sequencing analysis method is consistent with clinical effectiveness.
Clinically EGFR G719X is patient using targeted drug sensitivity site, and the ratio which mutates in patient population is 3% or so.EGFR T790X mutation is patient's issuable drug resistance secondary mutation during using targeted drug, is one of the reason of patient develops drug resistance.Abrupt climatic change that the sequencing analysis method obtains the result shows that, in the group being made of 223 peripheral blood from patients with lung cancer plasma samples, the frequency of mutation of EGFR T790X is that the frequency of mutation of 1.5%, EGFR G719X is 0.5%.Illustrate that the sequencing analysis method can detect the frequency of mutation ratio of extremely low frequency in peripheral blood blood plasma and Drug-resistant and sensitivity.
Clinically with the progress that targeted drug is treated, the increase of the gene copy numbers such as MET can cause patient to the drug resistance of targeted drug, and then disease progression occur.The copy number situation of change of MET gene of an example patients with lung cancer in the peripheral blood plasma DNA of different clinical stages disease progression I phases, disease progression II phase and disease progression III phase is had detected using the sequencing analysis method, the result shows that the copy number of the MET gene in the peripheral blood plasma DNA of this patients with lung cancer is continuously increased (Fig. 4) from the disease progression I phase to the disease progression III phase.Clinical diagnosis the result shows that, there is the case where to targeted drug drug resistance from the disease progression I phase to the disease progression III phase in this patients with lung cancer, and can explain from another point of view and detect the copy number increase extremely of the MET of cfDNA in the plasma sample of the disease progression III phase of this patients with lung cancer may be that patient one of the genome evidence of clinical drug-resistant symptom occurs.Illustrate that the sequencing analysis method can be well for the disease progression situation of the variation real-time tracking patient of copy number.
Patients with lung cancer ALK gene is merged with other genes and a kind of significant molecular diagnostic markers object of patients with lung cancer.It is found in the test of 15 peripheral blood from patients with lung cancer plasma samples of wheel using the sequencing analysis method, the detection frequency of ALK gene breaking point (breakpoint) is 93.3% (detection total number of samples is 15, and detection ALK gene breaking point sample number is 14).
Industrial application
It can directly reflect the gene mutation situation of tumor tissues in view of the gene mutation situation of ctDNA, the building of dissociative DNA sequencing library can be carried out using the construction method of dissociative DNA sequencing library of the invention, and then the ctDNA catastrophe of tumor patient and its variation of content are obtained by sequencing analysis, with early diagnosis detection, genetic heterozygosity assessment, tumour Monitoring on Dynamic Change, targeted therapy gene mutation analysis, tumour the early response to treatment evaluation, the detection of tumour minimal residue and drug resistance of tumor real-time detection etc. for carrying out tumour.

Claims (14)

  1. The construction method of dissociative DNA sequencing library, comprising:
    1) end reparation is carried out to dissociative DNA, obtains the dissociative DNA being repaired to end；It repairs and is carried out in the end reparation system containing archaeal dna polymerase and nucleoside monophosphate kinase in the end, concentration of the archaeal dna polymerase in the end reparation system is 1.5U-3U/50 μ L, and concentration of the nucleoside monophosphate kinase in the end reparation system is 5U-10U/50 μ L；
    2) end is carried out to the dissociative DNA that the end is repaired and add " A ", obtain the dissociative DNA that end adds " A "；The end adds " A " to add in A system and carry out in the end containing Klenow segment (3 ' -5 ' exo-), and the Klenow segment (3 ' -5 ' exo-) adds the concentration in " A " system to be 2.5U-5U/50 μ L in the end；
    3) the dissociative DNA linking library connector of " A " is added to obtain the dissociative DNA of belt lacing to the end；The linking library connector carries out in the adjunction head system containing library connector and DNA ligase, concentration of the library connector in the adjunction head system is 20-40pmol/50 μ L, and concentration of the DNA ligase in the adjunction head system is 1200U-2400U/50 μ L；
    4) dissociative DNA for expanding the belt lacing, obtains dissociative DNA library.
  2. Construction method according to claim 1, it is characterised in that: the volume that system is repaired in the end is 20-100 μ L.
  3. Construction method according to claim 1, it is characterised in that: the archaeal dna polymerase is T4DNA polymerase, and the nucleoside monophosphate kinase is T4 polynueleotide kinase, and the DNA ligase is T4DNA ligase.
  4. Construction method according to claim 1, it is characterized by: 4) amplification carries out in the amplification system containing Platinum Pfx archaeal dna polymerase, concentration of the Platinum Pfx archaeal dna polymerase in the amplification system is 2U-4U/50 μ L.
  5. Construction method according to claim 4, it is characterised in that: the circulating temperature program of the amplification is first 94 DEG C of 20s, then 62 DEG C of 40s, last 72 DEG C of 50s.
  6. Construction method according to claim 1, it is characterised in that: the construction method includes the steps that extracting the dissociative DNA from body fluid.
  7. Construction method according to claim 1, it is characterised in that: the dissociative DNA is the dissociative DNA in body fluid.
  8. Construction method according to claim 7, it is characterised in that: the body fluid is blood.
  9. Application of the construction method described in claim 1 in the sequencing of dissociative DNA.
  10. Application according to claim 9, it is characterised in that: the dissociative DNA is the dissociative DNA in body fluid.
  11. Application according to claim 10, it is characterised in that: the body fluid is blood.
  12. Application of the construction method described in claim 1 in detection dissociative DNA variation situation.
  13. Application according to claim 12, it is characterised in that: the dissociative DNA is the dissociative DNA in body fluid.
  14. Application according to claim 13, it is characterised in that: the body fluid is blood.
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CN107354148A (en) * 2017-08-17 2017-11-17 上海派森诺生物科技股份有限公司 A kind of method for efficiently building storehouse for minim DNA
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CN109957606A (en) * 2019-04-17 2019-07-02 杭州西合森医学检验实验室有限公司 Target the sequencing library construction method of medicine Resistance detection reagent
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020034161A (en) * 1999-07-19 2002-05-08 알. 씨. 제닝스 A method for amplifying low abundance nucleic acid sequences and means for performing said method
CN102127818A (en) * 2010-12-15 2011-07-20 张康 Method for creating fetus DNA library by utilizing peripheral blood of pregnant woman
CN103088433A (en) * 2011-11-02 2013-05-08 深圳华大基因科技有限公司 Construction method and application of genome-wide methylation high-throughput sequencing library and
CN103103624A (en) * 2011-11-15 2013-05-15 深圳华大基因科技有限公司 Method for establishing high-throughput sequencing library and application thereof
CN103361743A (en) * 2013-07-23 2013-10-23 安诺优达基因科技(北京)有限公司 DNA library construction method for prenatal diagnosis
CN103806111A (en) * 2012-11-15 2014-05-21 深圳华大基因科技有限公司 Construction method and application of high-throughout sequencing library

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020034161A (en) * 1999-07-19 2002-05-08 알. 씨. 제닝스 A method for amplifying low abundance nucleic acid sequences and means for performing said method
CN102127818A (en) * 2010-12-15 2011-07-20 张康 Method for creating fetus DNA library by utilizing peripheral blood of pregnant woman
CN103088433A (en) * 2011-11-02 2013-05-08 深圳华大基因科技有限公司 Construction method and application of genome-wide methylation high-throughput sequencing library and
CN103103624A (en) * 2011-11-15 2013-05-15 深圳华大基因科技有限公司 Method for establishing high-throughput sequencing library and application thereof
CN103806111A (en) * 2012-11-15 2014-05-21 深圳华大基因科技有限公司 Construction method and application of high-throughout sequencing library
CN103361743A (en) * 2013-07-23 2013-10-23 安诺优达基因科技(北京)有限公司 DNA library construction method for prenatal diagnosis

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