CN107541791A - Construction method, kit and the application in plasma DNA DNA methylation assay library - Google Patents
Construction method, kit and the application in plasma DNA DNA methylation assay library Download PDFInfo
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Abstract
The invention discloses a kind of construction method, kit and the application in plasma DNA DNA methylation assay library.Wherein, the construction method comprises the following steps：S1, plasma DNA is extracted from blood sample；S2, end reparation and 3 ' end addition A bases are carried out to plasma DNA；S3, the joint for modification that the end connection C of the obtained plasma DNAs of S2 is methylated；S4, the product obtained to S3 carry out bisulfite conversion, performing PCR of going forward side by side extension；S5, magnetic beads for purifying is carried out to S4 PCR primer, gets rid of the small fragment DNA and primer dimer of non-non-specific amplification；And S6, performing PCR amplification is entered to S5 product, and magnetic beads for purifying is carried out, obtain plasma DNA DNA methylation assay library.Apply the technical scheme of the present invention, reduce conventional trace methylation and build storehouse initial amount, simplify and build storehouse step and improve library organic efficiency, be to screen diagnosing tumor mark subsequently through plasma DNA methylation differential site to lay a good foundation.
Description
Technical field
The present invention relates to biological technical field, in particular to a kind of structure in plasma DNA DNA methylation assay library
Construction method, kit and application.
Background technology
Plasma free Circulating tumor DNA (Circulating tumor DNA, ctDNA) refers to by downright bad or apoptosis swollen
Oncocyte is discharged into the single-stranded or double-stranded DNA in blood plasma, accounts for the 0.1%-1% of dissociative DNA.CtDNA clip sizes are usual
For 160-180bp, half-life period 2 hours, carry SNP (point mutation), InDel (insertion and deletion), CNV (copies in blood
Number), fusion (fusion), the information such as methylate.CtDNA constantly flows in blood circulation system, can reflect in real time swollen
Knurl patient's current information.
Compared with traditional detection method, ctDNA detections have many advantages：1) sampling is convenient：CtDNA detections need to only draw blood i.e.
The parsing to DNA of tumor cell can be completed, the risk of tissue biopsy such as avoids puncturing, perform the operation.2) detection technique is ripe：Utilize two
CtDNA is detected for sequencing technologies (NGS), it is sensitive and accurate, false positive rate is low, it can detect as little as 0.1% low frequency mutation.3) examine
Survey comprehensive：For the releasable DNA of nearly all tumour cell into blood, ctDNA can embody the comprehensive condition of patient's body tumour.
For the foregoing reasons, ctDNA is expected to turn into the emerging biomarker of clinical detection, diagnosis and curative effect monitoring, accumulates
Containing huge clinical value.
DNA methylation refers to add a methyl group on 5C positions in the presence of DNA methylation enzyme in cytimidine,
Form 5-methylcytosine.Internal DNA methylation can cause the disease even generation of cancer extremely.DNA methylation detection tool
There are very strong stability and tissue specificity, recently research have indicated that the Non-invasive detection of prostate cancer can be realized by DNA methylation
(Yao L,Ren S,Zhang M,et al.Identification of specific DNA methylation sites
on the Y-chromosome as biomarker in prostate cancer[J].Oncotarget,2015,6(38):
40611.)。
CtDNA DNA methylation assays will have both the advantage of ctDNA detections and DNA methylation detection, and the diagnosis to tumour will have
There are higher sensitivity, specificity and diagnostic rate.
CtDNA DNA methylation assays are broadly divided into two classes：The detection of specific site and full-length genome DNA methylation assay.For spy
The detection technique of anchor point is based primarily upon PCR, such as：Standard PCR, methylation status of PTEN promoter, qRT-MSP etc..For full-length genome
DNA methylation assay, because ctDNA methylation sites concentration is too low, conventional BS-Seq, RRBS, MBD-Seq and MeDIP-Seq are
It is inapplicable.With the development of high throughput sequencing technologies, people gradually develop shotgun large-scale parallel sequencing, full-length genome first
(MCTA-Seq) technology is sequenced after base CpG Cascaded amplifications, for studying ctDNA full-length genome methylation level.
CfDNA contents are low (more than ten nanograms), therefore conventional trace methylation banking process (initial amount 30-100ng) is no
It is applicable.In prior art basis, inventor's Optimal Experimental parameter, the micro database technology that methylates suitable for cfDNA is explored,
Invention one is intended to based on the sequencing of two generations, the practicable banking process that methylates, for changing this research field cfDNA methyl
Change the predicament in terms of sequencing.
The content of the invention
The present invention is intended to provide a kind of construction method, kit and the application in plasma DNA DNA methylation assay library, with
The technical problem in conventional trace methylation banking process structure library can not be used by solving plasma DNA in the prior art.
To achieve these goals, according to an aspect of the invention, there is provided a kind of plasma DNA DNA methylation assay
The construction method in library.The construction method comprises the following steps：S1, plasma DNA is extracted from blood sample；S2, to blood
Starch dissociative DNA and carry out end reparation and 3 ' end addition A bases；S3, the end of the obtained plasma DNAs of S2 is connected into C methyl
Change the joint of modification；S4, the product obtained to S3 carry out bisulfite conversion, performing PCR of going forward side by side extension；S5, S4 PCR is produced
Thing carries out magnetic beads for purifying, gets rid of the small fragment DNA and primer dimer of non-non-specific amplification；And S6, to S5 product
Enter performing PCR amplification, and carry out magnetic beads for purifying, obtain plasma DNA DNA methylation assay library.
Further, the methylate joints of modification of C are deoxyribonucleoside as shown in SEQ ID NO.1 and SEQ ID NO.2
Acid sequence.
Further, blood sample is patients with prostate cancer blood sample.
Further, the step of construction method also includes carrying out quality testing to the library that structure obtains.
Further, the step of carrying out quality testing to library specifically includes：Library concentration is determined using Qubit, 2%
Agarose gel electrophoresis detection library DNA fragment distribution, if DNA fragmentation is distributed in 300bp or so in library, illustrates built text
Storehouse is that plasma DNA methylates library；Or library is built using Agilent2100/7500 qualitative and quantitative detections, if 2100/
7500 peak figure main peak integrated distributions are in 300bp or so, and without miscellaneous peak, then it is that plasma DNA methylates text that explanation, which builds library,
Storehouse；Or high-flux sequence is carried out using Illumina Hiseq3000, by bioinformatics means analysis data, assess library
Reads mapping rates and conversion ratio, if above parameter is respectively higher than 70.0%, 98.5%, then it is blood plasma that explanation, which builds library,
Dissociative DNA methylates library.
According to another aspect of the present invention, there is provided a kind of kit of plasma DNA DNA methylation assay library construction.
The kit includes：Plasma DNA extracts reagent, end reparation and the examination of 3 ' end addition A bases are carried out to plasma DNA
Agent, C methylate the joint of modification, joint connection reagent, bisulfite conversion reagent, magnetic beads for purifying is carried out to PCR primer
Reagent, get rid of the small fragment DNA of non-non-specific amplification and the reagent of primer dimer and the reagent for PCR amplifications.
Further, the methylate joints of modification of C are deoxyribonucleoside as shown in SEQ ID NO.1 and SEQ ID NO.2
Acid sequence.
In accordance with a further aspect of the present invention, there is provided a kind of plasma DNA DNA methylation assay text of construction method structure
Application of the storehouse in high-flux sequence.
Further, high-flux sequence is sequenced for Illumina platforms.
Further, plasma DNA comes from patients with prostate cancer blood sample.
Apply the technical scheme of the present invention, reduce conventional trace methylation and build storehouse initial amount, simplify and build storehouse step simultaneously
Library organic efficiency is improved, is to screen diagnosing tumor mark subsequently through plasma DNA methylation differential site to establish
Basis.
Brief description of the drawings
The Figure of description for forming the part of the application is used for providing a further understanding of the present invention, and of the invention shows
Meaning property embodiment and its illustrate be used for explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 shows the quality measurements that plasma DNA is extracted in embodiment 1；
Fig. 2 shows in embodiment 1 that plasma DNA methylates library agarose gel electrophoresis the qualitative analysis；
Fig. 3 shows in embodiment 1 that plasma DNA methylates library quantitative analysis results；
Fig. 4 shows in embodiment 1 that plasma DNA methylates libraries high throughput sequencing reads assessment result cake charts；
And
Fig. 5 shows in embodiment 1 that plasma DNA methylates libraries high throughput sequencing reads assessment results.
Embodiment
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the application can phase
Mutually combination.Describe the present invention in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
According to a kind of typical embodiment of the present invention, there is provided a kind of structure in plasma DNA DNA methylation assay library
Method.The construction method comprises the following steps：S1, plasma DNA is extracted from blood sample；S2, plasma DNA is entered
Row end is repaired and 3 ' end addition A bases；S3, the joint for modification that the end connection C of the obtained plasma DNAs of S2 is methylated
(design principle：F chain 5' ends phosphorylation, all C methylate modification, and all C of R chains methylate modification)；S4, the product obtained to S3
Carry out bisulfite conversion, performing PCR of going forward side by side extension；S5, magnetic beads for purifying is carried out to S4 PCR primer, got rid of not non-specific
Property amplification small fragment DNA and primer dimer；And S6, performing PCR amplification is entered to S5 product, and magnetic beads for purifying is carried out, obtain
To plasma DNA DNA methylation assay library.
Preferably, the methylate joints of modification of C are deoxynucleotide as shown in SEQ ID NO.1 and SEQ ID NO.2
Sequence, sequence are：SEQ ID NO.1(Multiplexing adapters-meth-F(5’->3’)：
GATCGGAAGAGCACACGTCT, 5 ' end phosphorylations, all C methylate modification；SEQ ID NO.2(Multiplexing
adapters-meth-R(5’->3’)：ACACTCTTTCCCTACACGACGCTCTTCCGATCT, all C methylate modification.
According to a kind of typical embodiment of the present invention, blood sample is patients with prostate cancer blood sample.
According to a kind of typical embodiment of the present invention, there is provided a kind of plasma DNA DNA methylation assay library construction
Kit.The kit includes：Plasma DNA extracts reagent, end reparation and 3 ' end addition A are carried out to plasma DNA
Reagent, the C of base methylate modification joint, joint connection reagent, bisulfite conversion reagent, to PCR primer carry out magnetic
The reagent of pearl purifying, get rid of the small fragment DNA of non-non-specific amplification and the reagent of primer dimer and expanded for PCR
Reagent.
Preferably, the methylate joints of modification of C are deoxynucleotide as shown in SEQ ID NO.1 and SEQ ID NO.2
Sequence.
According to a kind of typical embodiment of the present invention, there is provided a kind of plasma DNA first of above-mentioned construction method structure
Baseization detects application of the library in high-flux sequence.
Preferably, high-flux sequence is sequenced for Illumina platforms.
According to a kind of typical embodiment of the present invention, plasma DNA comes from patients with prostate cancer blood sample.
According to a kind of typical embodiment of the present invention, plasma DNA and first are identified by following at least three kinds of methods
Whether base library is qualified：
Authentication method one：
The library that the method that storehouse is built by a tubular type DNA methylation is built is subjected to Biology identification, is to utilize first
Qubit determines library concentration, 2% agarose gel electrophoresis detection library DNA fragment distribution, if DNA fragmentation is distributed in library
In 300bp or so, then it is that plasma DNA methylates library that explanation, which builds library,.
Authentication method two：
The library that the method that storehouse is built by a tubular type DNA methylation is built is subjected to Biology identification, utilized
Agilent2100/7500 qualitative and quantitative detections build library, if 2100/7500 peak figure main peak integrated distribution in 300bp or so,
And without miscellaneous peak, then it is that plasma DNA methylates library that explanation, which builds library,.
Authentication method three：
The library for the method structure for building storehouse by a tubular type DNA methylation respectively is subjected to Biology identification, utilized
Illumina Hiseq3000 carry out high-flux sequence, by bioinformatics means analysis data, assess library reads's
The parameters such as mapping rates, conversion ratio, if above parameter is respectively higher than 70.0%, 98.5%, then it is blood plasma trip that explanation, which builds library,
From DNA methylation library.
Beneficial effects of the present invention are further illustrated below in conjunction with example.
Embodiment 1
Major experimental step is as follows：
1. the acquisition of plasma sample
EDTA heparin tubes are used under typical laboratory conditions, preferably Streck heparin tubes collect peripheral blood of patients with prostate cancer
10ml, 1900g (or 3000rpm) room temperature Quick spin 10min, absorption upper plasma (avoiding leukocytic cream from polluting) gently arrive
New 15ml centrifuge tubes, 16000g room temperatures centrifugation 10min, gently draw supernatant to new 15ml centrifuge tubes.
2. the extraction and quality inspection of plasma DNA
Kai Jie companies are used to be used for the kit for extracting free nucleic acid under typical laboratory conditions, preferably
Circulating Nucleic Acid kits, extract dissociative DNA from the blood plasma being separated to specifications, and with 30 μ l
Warm water elutes, the plasma DNA extracted by the qualitative and quantitative detections of Agilent 2100, and testing result is as shown in figure 1, specific
Illustrated in table 1.
Table 1.Agilent 2100 detects cfDNA result explanations
Jian Ku and storehouse inspection 3. plasma DNA methylates
The plasma DNA of acquisition is built into the library that methylates according to following steps：
1) end-filling and plus A- tails
Using preferable NEB#E7442s enzymes, sample mixing (38.92 μ l systems) in the following proportions：End prep enzyme
Mix (1.80 μ l), End repair reaction buffer (3.89 μ l), Fragment DNA samples (30 μ l), λ DNA
(3.23μl).Centrifugation, 20 DEG C of incubations 30min, 65 DEG C of incubation 30min.
On the premise of other operation links are consistent, end reparation is attempted respectively plus (sample is successively completed in the modification of A tails
Beginning measures：31.75ng) and simultaneously complete (sample initial amount：26.64ng), PCR primer is respectively 3.96 μ g, 4.79 μ g, illustrates one
Footwork can effectively avoid sample loss.
2) joint connects
Using preferable NEB#E7445s ligases, sample mixing (50 μ l systems) in the following proportions：Blunt/7A ligase
Mix (8.98 μ l), NEB Next adapter (1.50 μ l), Ligation enhance (0.60 μ l), End-repair products
(38.92μl).Centrifugation, 20 DEG C of incubation 15min.In the present embodiment, joint sequence (adapters) used includes specific modification,
Sequence is：SEQ ID NO.1(Multiplexing adapters-meth-F(5’->3’))：
GATCGGAAGAGCACACGTCT, 5 ' end phosphorylations, all C methylate modification；SEQ ID NO.2(Multiplexing
adapters-meth-R(5’->3’))：ACACTCTTTCCCTACACGACGCTCTTCCGATCT, all C methylate modification.
3) bisulfite converts
DNA methylation conversion reagent box, preferably EZ DNA Methylation- are used under typical laboratory conditions
GoldTMJoint connection product is carried out C- by Kit (Zymo) kits>U is converted.In this experimentation, CT conversion
Reagent is formulated as 600 μ l ddH2O+300 μ l M-Dilution Buffer+50 μ l M-Dissolving Buffer, turn
It is 98 DEG C of incubations 10min, 64 DEG C of incubation 2h to change program.With 22 μ l warm water eluted dna samples, -20 DEG C freeze.
4) PCR amplifications 1
System (50 μ l)：2xKAPA mix(25μl),10μM primer 1.0(2.5μl),10μM index primer
2.0 (2.5 μ l), DNA sample (20 μ l).Program：98℃45s；98 DEG C of 15s, 65 DEG C of 30s, 72 DEG C of 30s；72℃60s；6 are followed
Ring.Amplimer is：Primer 1.0SEQ ID NO：3(5’->3’):AATGATACGGCGACCACCGAGATCTACACTCTTTC
CCTACACGACGCTCTTCCGATCT；Primer 2 .0SEQ ID NO：4(5’->3’):GTGACTGGAGTT
CAGACGTGTGCTCTTCCGATCT。
5) magnetic beads for purifying
1.2 × magnetic bead adsorption step 4) in sample, be stored at room temperature 5min, abandon supernatant.75% ethanol cleans 2 times, 22 μ l temperature
Water elution.
6) PCR amplifications 2 (condition primer is with PCR amplifications 1)
7) magnetic bead substep purifies
Large fragment first is adsorbed with 0.6 × magnetic bead, separates supernatant；0.25 × magnetic bead adsorbs foregoing supernatant, stands 5min and abandons
Clearly；75% ethanol is washed twice, and the elution of 22 μ l warm water, -20 DEG C freeze.
After cfDNA libraries PCR primer is obtained, glue reclaim method and the recovery of substep paramagnetic particle method, organic efficiency point is respectively adopted
Not Wei 7.8%, 34.9%, the latter is approximately the former 4.5 times.
8) storehouse is examined
DNA sample (see Fig. 2,3) in 2% agarose gel electrophoresis, Agilent2100/7500 qualitative, quantitatives 7, specifically
It is bright to be shown in Table 2.
Table 2.Agilent 2100 detects cfDNA detection library result explanations
4. plasma DNA methylates, libraries high throughput is sequenced
Library will be built in step 3 and carries out high-flux sequence using Illumina platforms, DNA methylation library sample during upper machine
Product and DNA library survey 30G data with 50% ratio sample mixing, each sample, and bioinformatic analysis surveys reads mass, as a result
Display：Single-ended reads mapping rates are higher than 70%, C->U high conversion rates are in 98.5% (see Fig. 4,5).Assessment parameter meets pre-
Phase, illustrate that built library is qualified.
As can be seen from the above description, the above embodiments of the present invention realize following technique effect：
1) traditional DNA methylation banking process needs to interrupt genomic DNA by physical method, easily causes DNA
Fragment is damaged, and the present invention directly extracts plasma DNA, it is not necessary to carries out the processing that traditional DNA sample interrupts, avoids DNA people
The hereditary information carried for damage, high-fidelity DNA；
2) traditional minigene group DNA methylation banking process sample initial amount is 30~100ng, and the present invention uses one
Since tubular type (expand, all reactions are all implemented in an EP pipe, reduce the sample caused by tube wall adsorbs i.e. building storehouse to PCR
Product lose) reduce and build storehouse initial amount, preferably initial amount as little as 10ng；
3) end is repaired and adds the step of A tails one to complete during building storehouse, reduces sample loss；
4) joint sequence used in this method (adapters) includes specific modification, and F chains 5 ' hold phosphorylation, institute on F chains and R chains
There is C to methylate modification；
5) glue reclaim method purified pcr product is replaced using substep magnetic bead absorption method, improves product recovery efficiency.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Sequence table
<110>Beijing Institute of Gene Science, Chinese Academy of Sciences
<120>Construction method, kit and the application in plasma DNA DNA methylation assay library
<141> 2017-10-26
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Claims (10)
1. a kind of construction method in plasma DNA DNA methylation assay library, it is characterised in that comprise the following steps：
S1, plasma DNA is extracted from blood sample；
S2, end reparation and 3 ' end addition A bases are carried out to the plasma DNA；
S3, the joint for modification that the end connection C of the obtained plasma DNAs of the S2 is methylated；
S4, the product obtained to the S3 carry out bisulfite conversion, performing PCR of going forward side by side extension；
S5, magnetic beads for purifying is carried out to the PCR primer of the S4, gets rid of the small fragment DNA and primer two of non-non-specific amplification
Aggressiveness；And
S6, performing PCR amplification is entered to the product of the S5, and carry out magnetic beads for purifying, obtain the plasma DNA DNA methylation assay
Library.
2. construction method according to claim 1, it is characterised in that the methylate joints of modification of the C are such as SEQ ID
Deoxynucleotide sequence shown in NO.1 and SEQ ID NO.2.
3. construction method according to claim 1, it is characterised in that the blood sample is patients with prostate cancer blood sample
This.
4. construction method according to claim 1, it is characterised in that the construction method also includes the text obtained to structure
Storehouse carries out the step of quality testing.
5. construction method according to claim 4, it is characterised in that described the step of carrying out quality testing to library is specific
Including：
Library concentration, 2% agarose gel electrophoresis detection library DNA fragment distribution, if DNA in library are determined using Qubit
Fragment is distributed in 300bp or so, then it is that plasma DNA methylates library that explanation, which builds library,；Or
Library is built using Agilent2100/7500 qualitative and quantitative detections, if 2100/7500 peak figure main peak integrated distribution exists
300bp or so, and without miscellaneous peak, then it is that plasma DNA methylates library that explanation, which builds library,；Or
High-flux sequence is carried out using Illumina Hiseq3000, by bioinformatics means analysis data, assesses library
Reads mapping rates and conversion ratio, if above parameter is respectively higher than 70.0%, 98.5%, then it is blood plasma that explanation, which builds library,
Dissociative DNA methylates library.
A kind of 6. kit of plasma DNA DNA methylation assay library construction, it is characterised in that including：Plasma DNA carries
Take reagent, end reparation carried out to plasma DNA and 3 ' ends are added reagent, the C of A bases and methylated joint, the joint of modification
Reagent, bisulfite conversion reagent, the reagent that magnetic beads for purifying is carried out to PCR primer are connected, gets rid of non-non-specific amplification
Small fragment DNA and primer dimer reagent and for PCR amplification reagent.
7. kit according to claim 1, it is characterised in that the methylate joints of modification of the C are such as SEQ ID
Deoxynucleotide sequence shown in NO.1 and SEQ ID NO.2.
8. a kind of plasma DNA DNA methylation assay library of the structure of the construction method as any one of claim 1 to 5 exists
Application in high-flux sequence.
9. application according to claim 8, it is characterised in that the high-flux sequence is sequenced for Illumina platforms.
10. application according to claim 8, it is characterised in that the plasma DNA comes from patients with prostate cancer blood
Sample.
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| CN114606316A (en) * | 2022-03-12 | 2022-06-10 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Construction method of model for early diagnosis and prognosis prediction of NK/T cell lymphoma |
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