CN106893774A - The method that DNA Deflection levels are detected with polymolecular label - Google Patents
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Abstract
The present invention relates to a kind of method that use polymolecular label detects DNA Deflection levels, the first DNA molecular label N is inserted in D51, obtain the first single strand dna, wherein N1Sequence length is 2 12bp；The second DNA molecular label N is inserted in D72With the 3rd DNA molecular label N3, obtain the second single strand dna, N2And N3Sequence length is respectively 2 12bp, 2 20bp；By above-mentioned two single chain molecules mixing after annealing, then DNA polymerisations are carried out, then carry out digestion using restriction enzyme, obtain the DNA joint sequences containing the first cohesive end；DNA to be measured is broken into DNA short-movie sections, and at its two ends plus the second cohesive end with the first cohesive end reverse complemental；Connection DNA joint sequences and DNA short-movie sections, are sequenced after PCR amplifications to it, are then compared with mankind's reference gene group, to judge DNA Deflection levels to be measured.
Description
Technical field
The side of DNA Deflection levels is detected the present invention relates to DNA variation detection fields, more particularly to a kind of use polymolecular label
Method.
Background technology
Liquid Biopsy, compared to traditional detection method, because with Small side effects, simple to operate, energy repeated sampling
The features such as, it is the very powerful and exceedingly arrogant technology of the accurate medical field of current tumour, it predominantly detects object includes：Circulating tumor cell
(CTC), Circulating tumor DNA (ctDNA) and tumour excretion body etc., can point out the information such as tumor development process and the resistance to the action of a drug,
The medication of individuation is instructed to treat.
CtDNA be it is a kind of be present in the body fluid such as blood plasma or serum, cerebrospinal fluid it is extracellular free single-stranded or double-stranded
DNA, mostlys come from necrosis or the tumour cell of apoptosis, the efflux body of tumor cell secretion and circulating tumor cell.CtDNA pieces
Section is typically sized to 160-180bp, in blood half-life period 2 hours, carries point mutation, insertion and deletion, copy number, fusion base
Because waiting abrupt information, constantly flowed in blood circulation of human body system, can in real time reflect the current information of tumor patient.However,
Early stage most of solid tumor and part entity knurl middle and advanced stage, the ctDNA contents in blood plasma are extremely low, using high-flux sequence
The effective data rate that technology (NGS) is obtained is relatively low, at the same it is higher to requirement of experiment, limit experiment sample consumption and experiment number.
Secondly contain substantial amounts of genomic DNA in ctDNA samples, cause high-flux sequence ambient noise very high, be sequenced with conventional storehouse of building
Cause tumor signal to be submerged in ambient noise completely during method, the detection of ctDNA can only be improved by increasing sequencing depth
Accuracy, this not only adds sequencing cost, while inevitably there is the amplification mistake that PCR in experimentation is introduced.
In due to the sequencing procedure when library construction, fasciation are into and while synthesis, there is PCR (PCR) polymerizations
The amplification mistake of enzyme, causes NGS that each base error rate is sequenced 5 × 10-4To 10-2.Therefore, the variation less than the frequency is examined
Survey will be unable to be distinguished with experimental error.
At present, a kind of to be referred to as UID (unique mark) technology, also referred to as molecular label technology is used for NGS error correctings.
The technology is that every molecule is introduced into a recognizable only sequential coding (barcode) before PCR amplifications, by this only
Come which is distinguished is molecular sequences that pcr amplification primer enters, which is original molecular sequences in sample, so as to carry for one sequential coding
Sequencing data high is efficient；Real variation and experimental error can also be distinguished by the only sequential coding simultaneously, to carry
Height sequencing accuracy, the especially low frequency to ctDNA samples are mutated, significant.
And whether current UID technologies can't distinguish the extension increasing sequence from same double-stranded DNA template while containing
Two chains of the double-strand；And prior art prepares barcode using completely synthetic method, cost is very high, Er Qieqi
Barcode length is not optimal；Accurately mate is required when barcode compares in prior art analysis method, but is sequenced because existing
Mistake, same barcode there may be part variation in sequencing, cause data to waste.
The content of the invention
In order to solve the above technical problems, detecting DNA Deflection levels with polymolecular label it is an object of the invention to provide one kind
Method, before PCR amplifications on double-stranded DNA introduce two kinds of molecular labels to distinguish two chains of DNA, improve DNA and make a variation
The accuracy of detection.
The invention provides a kind of method that use polymolecular label detects DNA Deflection levels, comprise the following steps：
(1) the first DNA molecular label N is inserted in single strand dna D51, obtain first single stranded DNA point of following sequence
Son：
5'-AATGATACGGCGACCACCGAGATCTACACN1ACACTCTTTCCCTACACGACGCTCTTCC GATCT-
3', wherein N1Sequence length is 2-12bp；
(2) the second DNA molecular label N is inserted in single strand dna D72With the 3rd DNA molecular label N3, obtain as follows
Second single strand dna of sequence：
5'-TCTTCTACAGTCAN2AGATCGGAAGAGCACACGTCTGAACTCCAGTCACN3ATCT
CGTATGCCGTCTTCTGCTTG-3', wherein N2Sequence length is 2-12bp, N3Sequence length is 2-20bp；
(3) there is annealing after mixing the first single strand dna in step (1) and (2) and the second single strand dna anti-
Should, make that first, second single strand dna is complementary to be combined, form Y type DNA moleculars, then in the presence of archaeal dna polymerase with it is de-
There is DNA polymerisations in oxygen ribonucleotide triphosphate (dNTP), make the 3 ' of the first single strand dna sequence to hold and extend, with second
5 ' end reverse complementals of single strand dna, then carry out digestion using restriction enzyme, obtain containing the first cohesive end
DNA joint sequences；
(4) DNA to be measured is broken into DNA short-movie sections, with archaeal dna polymerase at DNA short-movie sections two ends plus the second viscosity end
End, the second cohesive end and the first cohesive end reverse complemental；
(5) the DNA short-movie sections that the DNA joint sequences obtained with DNA ligase Connection Step (3) are obtained with step (4), so
Laggard performing PCR amplification, obtains amplified production；
(6) amplified production is sequenced, then carries out sequence alignment with mankind's reference gene group, to judge DNA to be measured
Deflection level.
Further, in step (1) and step (2), N1Sequence, N2Sequence and N3Sequence is random sequence, in each sequence
Base be randomly selected from A, T, C and G.
Further, in step (3), the condition of annealing reaction is：95 DEG C, 10min；70 DEG C, 10min；65 DEG C,
10min；60 DEG C, 10min；55 DEG C, 10min；50 DEG C, 10min；25 DEG C, 10min terminates；Each circulation rate of temperature fall be
5%.
Further, in step (3), archaeal dna polymerase is Klenow enzymes or TAQ archaeal dna polymerases.
Further, in step (3), the reaction temperature of DNA polymerisations is 37 DEG C, and the reaction time is 1h.
Further, in step (3), following components is included during DNA polymerisations：Y type DNA molecular joints, 12.5 μ L；
The μ L of No. 2 buffer solutions of NEB boards 5 of 10 times of dilution；The μ of mixing deoxyribonucleoside triphosphate 2.5 of the TAKARA boards of concentration 2.5mM/L
L；DNA polymerase activity is the Klenow fragments without 5 prime excision enzyme activity of 5U/ μ L, 2.5 μ L；Seedless sour water, 27.5 μ L.
Further, in step (3), restriction enzyme is HpyCH4III, the identification sequence of the restriction enzyme
It is classified as ACAGT.
Further, in step (3), restriction enzyme is Fnu4HI, the recognition sequence of the restriction enzyme
It is GCTGC.
Further, in step (4), archaeal dna polymerase is Klenow enzymes.
Further, in step (4), the length of DNA short-movie sections is 150-300bp.
Further, in step (5), DNA ligase is T4DNA ligases or E.coli DNA ligases.
The present invention extends to the length of the molecular label of Y types DNA molecular in the prior art, implementation method and existing conjunction
It is identical into Y-shaped connector method, and the method for existing pre-synthesis molecular label is relatively costly, the present invention builds the PCR mistakes in storehouse using DNA
The method addition that base complementrity extends in journey is got on.
Further, in step (6), both-end sequencing or single-ended sequencing are carried out to amplified production.
Further, in step (6), a small number of base mispairings, the first DNA molecular label N are allowed during comparison1With second
DNA molecular label N2Random sequence is the same from DNA sequence dna from same template dna molecule.The ratio of analysing amplified product
To result, if all there is same variation on the DNA sequence dna of same template dna molecule, the variation is DNA to be measured
Middle occurred real DNA variations.
Using the method for the present invention, DNA sequence dna is compared to mankind's reference gene group, DNA fragmentation is in reference gene group
The Origin And Destination identical sequence of position be divided into one group, relatively with the first DNA molecular label N at group DNA sequence dna two ends1Sequence
Row, in the case of allowing a base mispairing, the first DNA molecular label N1Sequence identical DNA sequence dna is thought to come from same
DNA double chain, compares its second DNA molecular label N in these DNA sequence dnas2, in the case where a base mispairing is allowed, such as
There are two kinds of second different DNA molecular label N in fruit2, then it is assumed that the double-strand of DNA is detected simultaneously by, has all been existed in double-strand
Variation is real DNA variations.Because having connect the molecular label of random sequence in DNA double chain respectively, these molecular labels are almost
Will not be the same, so the molecular label carried in many sequences from same template is not quite identical, in this way
The extension increasing sequence that different templates are obtained can be distinguished, when then comparing again, the accuracy of DNA variation detections can be improved.
By such scheme, the present invention at least has advantages below：
The present invention has used two kinds of molecular labels to distinguish the DNA double chain after amplification, the change existed in double-strand simultaneously
Different is real DNA variations, improves the accuracy of DNA variation detections；In the NGS detection methods of existing ctDNA, addition point
One times more than the cost without molecular label method, the present invention is matched somebody with somebody the cost of subtab method by the base during PCR
To method molecular label of the addition with random sequence for extending, the cost of addition molecular label in NGS sequencings is effectively reduced,
Its cost, only than many 1/3 without molecular label, is that being widely popularized for molecular label technology reduces threshold.
Brief description of the drawings
Fig. 1 is the schematic diagram of the Y type DNA moleculars before 3 ' the non-polishings in end of the first DNA molecular sequence of the invention；
Fig. 2 is the DNA joint schematic diagrames of the cohesive end of band first formed after digestion；
Fig. 3 is the DNA short-movie section schematic diagrames for having added the second cohesive end；
Fig. 4 is the schematic diagram after DNA joint sequences are connected with DNA short-movie sections.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiment of the invention is described in further detail.Hereinafter implement
Example is not limited to the scope of the present invention for illustrating the present invention.
Embodiment 1
1st, the synthesis of joint (adapter) and annealing
1) the following sequence in synthesis table 1, it is a DNA of 8bp that the NNNNNNNN in wherein J-AdD5-8N represents length
Molecular label N1, in J-AdD703-10MMMMMMMMMMMIt is the second DNA molecular label N of 10bp to represent length2, wherein the
Three DNA molecular label N3It is pre-synthesis short-movie section, the overstriking base sequence CGCTCATT as in the sequence.
Table 1
2) centrifugation makes Adapter powder be gathered in bottom of the tube.
3) it is careful to open Adapter lids, it is careful not to allow powder to blow out.Respectively to J-AdD5-8N, J-AdD703-10M
The OAB dissolved powders of middle addition respective volume, make final concentration of 100 μM, and -20 DEG C preserve the storing liquid, standby.OAB Buffer
(oligomerization annealing buffer), composition is as shown in table 2：
Table 2
4) 25 μ L J-AdD5-8N (100 μM) are taken to be well mixed with 25 μ L J-AdD703-10M (100 μM), 54 μ L are obtained
Concentration is 50 μM of mother liquor, is placed in the PCR pipe of 0.1mL, mark Annealed Adapter.
5) above-mentioned two complementary element is combined using the annealing process in such as table 3 on ABI 9700PCR instrument, forms Y types
DNA molecular, the mother liquor after annealing is placed in -20 DEG C of preservations, further according to needing to be diluted to working solution concentration when taking.
Table 3
2nd, joint Quality Control after annealing
The Adapter for having annealed is extracted a part and is diluted to 1 μM, detected using the reagents of Agilent 2100DNA 1000
3rd, connector end filling-in
Using 50 μ L reaction systems in such as table 4,1h, 4 DEG C of holdings are incubated at 37 DEG C in the PCR instrument.
Table 4
| Component | Volume (μ L) | Final concentration |
| Annealed Adapter | 12.5 | |
| 10x NEB Buffer#2 | 5 | 1x |
| TAKARA dNTP mix(2.5mM each) | 2.5 | |
| Klenow exo-(5U/μl) | 2.5 | 12.5U |
| Nuclease-Free water | 27.5 | |
| Total | 50 |
4th, the joint after filling-in is purified using ethanol precipitation methods
The sodium acetate and 2.5 times of absolute ethyl alcohols of volume of 1/10 volume are added, -80 DEG C of placement 30min to 2h after mixing.So
Afterwards in 4 DEG C of centrifugations, 16000rcf centrifugations 30min；After abandoning supernatant, plus 750 μ L freeze 80% ethanol, 4 DEG C of centrifugation 16000rcf
5min；Supernatant is discarded, brief centrifugation to 1000rcf is stopped by stop；Blot room temperature after liquid and uncap and dry 5min；Plus 50 μ L
Nuclease free water, thorough dissolution precipitation mixes centrifugation, and brief centrifugation to 1000rcf is stopped by stop, marks Extended
Adapter, is stored in -20 DEG C.After taking 25 times of 1 μ l dilutions, the quality inspections of Agilent 2100DNA 1000 are carried out.
5th, joint digestion
Using NEB HpyCH4III restriction endonucleases, 37 DEG C of incubation 16h, 4 DEG C of holdings, reaction system such as table on 8800PCR instrument
Shown in 5：
Table 5
| Component | Volume (μ L) | Final concentration |
| Extended Adapter | 15 | |
| 10x NEB CutSmart Buffer | 5 | 1x |
| HpyCH4III(5U/μl) | 5 | |
| ddH2O | 25 | |
| Total | 50 |
6th, the joint after digestion is purified using ethanol precipitation methods
The sodium acetate and 2.5 times of absolute ethyl alcohols of volume of 1/10 volume are added, -80 DEG C of placement 30min to 2h after mixing.So
Afterwards in 4 DEG C of centrifugations, 16000rcf centrifugations 30min；After abandoning supernatant, plus 750 μ L freeze 80% ethanol, 4 DEG C of centrifugation 16000rcf
5min；Supernatant is discarded, brief centrifugation to 1000rcf is stopped by stop；Blot room temperature after liquid and uncap and dry 5min；Plus 30 μ L
Nuclease free water, thorough dissolution precipitation mixes centrifugation, and brief centrifugation to 1000rcf is stopped by stop, marks Extended
Adapter, is stored in -20 DEG C.After taking 10 times of 1 μ l dilutions, the quality inspections of Agilent 2100DNA 1000 are carried out.
Sequencing library is built using Roche Hyper banking process, joint is 10 with the molar ratio of DNA:1, experiment step
It is rapid as shown in table 6.
Table 6
After improved joint is prepared, DNA is carried out to build storehouse by Illumina routine banking process, will be to be detected
DNA is broken into the DNA short-movie sections that length is 200bp with the broken instrument of DNA, with E.coli DNA ligases connect DNA short-movie sections with
The adapter of transformation, obtains DNA connection products；The DNA connection products that PCR amplifications are obtained, obtain amplified production；With
Illumina Hiseq4000 sequenators carry out both-end sequencing to amplified production.
Then sequencing result is compared to mankind's reference gene group, DNA fragmentation with the comparison software bwa based on bwt algorithms
The Origin And Destination identical sequence of the position in reference gene group is divided into one group, relatively with the first of group DNA sequence dna two ends
DNA molecular label N1Sequence, in the case of allowing a base mispairing, the first DNA molecular label N1Sequence identical DNA sequence dna
Think to come from same DNA double chain, its second DNA molecular label N is compared in these DNA sequence dnas2, allowing a base mistake
In the case of matching somebody with somebody, if there is two kinds of second different DNA molecular label N2, then it is assumed that the double-strand of DNA has been detected simultaneously by, it is double
The variation all existed on chain is real DNA variations.
Original method molecular label is all synthesis, if the first DNA molecular tag length is 4bp, the second DNA molecular
Tag length 2, then will will then be respectively synthesized 4096 kinds of joints, and cost is very high.The first DNA molecular label of this method and second
DNA molecular label uses the method for disposable random synthesis, low cost to make simple.In sequencing, if length is 6bp alkali
2% is vicious in second DNA molecular label of base, after allowing 1 base mispairing, can save this 2% data.The present invention
Whether multiple copies of same DNA profiling generation in DNA cloning are recognized with increased 2 kinds of molecular labels and have DNA's simultaneously
Double-strand.
Fig. 1 is the schematic diagram of the Y type DNA moleculars before 3 ' the non-polishings in end of the first DNA molecular sequence of the invention, wherein length
For the NNNNNNNN of 8bp represents the first DNA molecular label, length represents the second DNA molecular label for the MMMMMMMMMM of 10bp,
TTACTCGC in D7 represents the 3rd DNA molecular label.
Fig. 2 is the DNA joint schematic diagrames of the cohesive end of band first formed after digestion, is formed wherein after digestion and carries base
First cohesive end of T.Fig. 3 is the DNA short-movie section schematic diagrames for having added the second cohesive end, and the second cohesive end is A.
Fig. 4 is the schematic diagram after DNA joint sequences are connected with DNA short-movie sections.The DNA joint sequences of Fig. 2 randomly with Fig. 3
DNA short-movie sections two ends combine.
Embodiment 2
In the synthesis of the joint (adapter) in the 1st step and annealing reaction, the following sequence in synthesis table 7, wherein J-
It is the first DNA molecular label N of 8bp that CGCTCATT in AdD5 represents length1, in J-AdD703-8N-10NNNN NNN NNNNIt is the second DNA molecular label N of 10bp to represent length2, it is the 3rd DNA molecular label of 8bp that NNNNNNNN represents length
N3, the DNA sequence dna of synthesis is as follows：
Remaining step is same as Example 1.
Embodiment 3
In the synthesis of the joint (adapter) in the 1st step and annealing reaction, the following sequence in synthesis table 8, wherein J-
It is the first DNA molecular label N of 6bp that NNNNNN in AdD5-6N represents length1, the NNNNNN representative length in J-AdD703-6N
Spend the second DNA molecular label N for 6bp2, wherein the 3rd DNA molecular label N3It is pre-synthesis short-movie section, the as sequence
In overstriking base sequence CGCTCATT.
Table 8
Remaining later step is same as Example 1.The first DNA molecular label N in various embodiments above1Sequence,
Two DNA molecular label N2Sequence and the 3rd DNA molecular label N3Base in sequence is randomly selected from A, T, C and G.
The above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, on the premise of the technology of the present invention principle is not departed from, can also make it is some improvement and
Modification, these are improved and modification also should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of method that use polymolecular label detects DNA Deflection levels, it is characterised in that comprise the following steps：
(1) the first DNA molecular label N is inserted in single strand dna D51, obtain the first single strand dna of following sequence：
5'-AATGATACGGCGACCACCGAGATCTACACN1ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', its
Middle N1Sequence length is 2-12bp；
(2) the second DNA molecular label N is inserted in single strand dna D72With the 3rd DNA molecular label N3, obtain following sequence
The second single strand dna：
5'-TCTTCTACAGTCAN2AGATCGGAAGAGCACACGTCTGAACTCCAGTCACN3ATCTCGTATGCCGTCTTCTG
CTTG-3', wherein N2Sequence length is 2-12bp, N3Sequence length is 2-20bp；
(3) there is annealing reaction after mixing the first single strand dna in step (1) and (2) and the second single strand dna,
Make that first, second single strand dna is complementary to be combined, form Y type DNA moleculars, then in the presence of archaeal dna polymerase with deoxidation core
There is DNA polymerisations in riboside triphosphoric acid, make the 3 ' of the first single strand dna sequence to hold and extend, with the second single strand dna
5 ' end reverse complementals, then carry out digestion using restriction enzyme, obtain the DNA joint sequences containing the first cohesive end；
(4) DNA to be measured is broken into DNA short-movie sections, with archaeal dna polymerase at the DNA short-movie sections two ends plus the second viscosity end
End, second cohesive end and the first cohesive end reverse complemental；
(5) the DNA short-movie sections that the DNA joint sequences obtained with DNA ligase Connection Step (3) are obtained with step (4), Ran Houjin
Performing PCR is expanded, and obtains amplified production；
(6) amplified production is sequenced, then carries out sequence alignment with mankind's reference gene group, to judge DNA to be measured
Deflection level.
2. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that in step
(3) in, the condition of the annealing reaction is：95 DEG C, 10min；70 DEG C, 10min；65 DEG C, 10min；60 DEG C, 10min；55 DEG C,
10min；50 DEG C, 10min；25 DEG C, 10min terminates；The rate of temperature fall of each circulation is 5%.
3. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that：In step
(3) in, the archaeal dna polymerase is Klenow enzymes or TAQ archaeal dna polymerases.
4. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that：In step
(3) in, the reaction temperature of the DNA polymerisations is 37 DEG C.
5. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that：In step
(3) in, the restriction enzyme is HpyCH4III, and the recognition sequence of the restriction enzyme is ACAGT.
6. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that：In step
(3) in, the restriction enzyme is Fnu4HI, and the recognition sequence of the restriction enzyme is GCTGC.
7. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that：In step
(4) in, the archaeal dna polymerase is Klenow enzymes.
8. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that：In step
(4) in, the length of the DNA short-movie sections is 150-300bp.
9. the method that use polymolecular label according to claim 1 detects DNA Deflection levels, it is characterised in that：In step
(5) in, the DNA ligase is T4DNA ligases or E.coli DNA ligases.
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| US11286524B2 (en) | 2016-08-29 | 2022-03-29 | Amoy Diagnostics Co., Ltd. | Multi-position double-tag connector set for detecting gene mutation and preparation method therefor and application thereof |
| CN110491445A (en) * | 2018-05-11 | 2019-11-22 | 广州华大基因医学检验所有限公司 | UID sequencing, UID sequence design, the method and application of the correction of UID duplicate removal mass value |
| CN110491445B (en) * | 2018-05-11 | 2023-05-30 | 广州华大基因医学检验所有限公司 | UID sequencing, UID sequence design, UID duplicate removal quality value correction method and application |
| CN108804873A (en) * | 2018-06-29 | 2018-11-13 | 首度生物科技（苏州）有限公司 | The device of Non-invasive detection parent and embryo genetic exception |
| CN109576347A (en) * | 2018-12-06 | 2019-04-05 | 深圳海普洛斯医疗器械有限公司 | The sequence measuring joints of the label containing unimolecule and the construction method of sequencing library |
| CN110129415A (en) * | 2019-05-17 | 2019-08-16 | 凯杰（苏州）转化医学研究有限公司 | A kind of NGS builds library molecular adaptor and its preparation method and application |
| CN110129415B (en) * | 2019-05-17 | 2023-08-18 | 迈杰转化医学研究(苏州)有限公司 | NGS library-building molecular joint and preparation method and application thereof |
| CN111073961A (en) * | 2019-12-20 | 2020-04-28 | 苏州赛美科基因科技有限公司 | High-throughput detection method for gene rare mutation |
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