CN108165610A - A kind of unicellular whole genome amplification kit - Google Patents
A kind of unicellular whole genome amplification kit Download PDFInfo
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- CN108165610A CN108165610A CN201711405983.7A CN201711405983A CN108165610A CN 108165610 A CN108165610 A CN 108165610A CN 201711405983 A CN201711405983 A CN 201711405983A CN 108165610 A CN108165610 A CN 108165610A
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- archaeal dna
- primer
- dna polymerase
- whole genome
- unicellular
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The present invention provides a kind of kit for being more preferably suitable for unicellular whole genome amplification, is on the one hand intended to through optimizational primer and amplification step, can be efficiently applied to the unicellular sample after fixing；It can be applied to a kind of in unicellular sample amplification with the enzymatic treatment of specific excision uridylate simultaneously, the error rate of amplification can be significantly reduced.Relative to the prior art, the kit is more efficient, simple, practical, accurate, loss less, at low cost, method is reproducible, error rate is low, is very suitable for the unicellular whole genome amplification kit of fixed sample, sample application range is expanded, error rate is reduced simultaneously, improves the precision of detection.
Description
Technical field
This patent belongs to field of biology, is related to a kind of unicellular whole genome amplification kit more particularly to a kind of spy
Not Shi He fixed sample unicellular whole genome amplification, this patent has expanded sample application range, while reduces error rate,
The precision of detection is improved, therefore is with a wide range of applications.
Background technology
High throughput sequencing technologies are fast-developing, most of sequencing sample used at present or millions of even more cells
Sample.Since cell number is numerous, the result studied it is the average value of signal or only in a group cell
Represent the cellular informatics of wherein preponderance, and individual cells it is exclusive characteristic it is often ignored.Particularly ground in tumour
In studying carefully, gene mutation or copy number variation are existed only in only a few cell (such as early stage cancer cell), and many cells are expanded and divided
Analysis can not detect these small variations, and it is particularly important that this allows for unicellular sequencing.
With the development of technology, unicellular sequencing is increasingly favored by researcher, and unicellular sequencing technologies are just gradual
Become the clinical tool of guidance from experiment research, based on study to clinic conversion and built bridge.It is slender at present
Born of the same parents' amplification method mainly has degenerate oligonucleotide primed PCR to expand (Degenerated Oligonucleotide Primed
PCR, DOP-PCR), multiple displacement amplification (Multiple Displacement Amplification, MDA) and repeatedly move back
Fire ring shape cyclic amplification (Multipe Annealing and Looping-based Amplification Cycles,
MALBAC) etc..But in the prior art, most of amplification methods are only applicable to living cells sample, and to fixed by formaldehyde
Sample amplification efficiency is poor, strongly limits single celled application range, how to solve fixed sample and be applicable in have become existing skill
One of problem to be resolved in art.
In order to solve this problem, this patent provide kit in sample cracking process by adjusting agent formulations,
It adds in the modes such as solution cross-linking step, joint magnetic bead PCR to optimize, is applicable to the amplification of unicellular sample after fixing, so as to
So that amplification object is not only only applicable to living cells sample, fixed sample is applicable to, expands use scope, had more
It is widely applied prospect.
Base mistake is also one of main bugbear faced in the unicellular amplification procedure of the prior art simultaneously.Numerous studies refer to
Go out, the false positive of SNV is mainly that cytimidine is mutated into thymidine (C → T), if it is possible to reduce the amount of C → T mutation, so that it may
The base error rate in unicellular amplification procedure is greatly lowered, the false positive of SNV is reduced.The prior art is in amplification procedure
In, it is larger to obtain high-fidelity, high coverage genomic information difficulty, other than improving amplification method and analysis means, sample
The cracking of product is often ignored.In cracking process, genomic DNA uncoiling is into linear DNA double-strand, at the same time, part
Cytimidine meeting deamination becomes uracil.Although this process can also occur once in a while in the cell, when cracking in vitro, mutation
Frequency then greatly improve.If the uracil of these mutation can be amplified in exponential amplification, eventually lead to without reparation
SNV false positives.In practical operation, cleavage method, the fidelity of archaeal dna polymerase, amplification region G/C content etc. can all lead to alkali
Base mistake, so as to generate non-genuine single nucleotide variations, i.e. SNV false positive issues.
Therefore in order to solve the base error rate occurred in unicellular amplification is high, amplification bias amount is big, amplification poor quality,
The problems such as false positive rate is high, this patent creatively design simultaneously and add in enzymatic treatment step, it is unexpected can be with by one kind
The enzyme of specific excision uridylate is applied in unicellular sample amplification method, can significantly reduce the mistake of amplification
Rate reduces the single nucleotide variations for knowing clearly non-genuine and generates, while utilize parallel samples analytical plan, solves that may be present etc.
Position gene delection problem.
In brief, this patent provide it is a kind of preferably efficiently, it is simple, practical, precisely, loss less, at low cost, method
It is reproducible, error rate is low, be very suitable for fixed sample application unicellular whole genome amplification kit, expanded sample
Application range, while error rate is reduced, improve the precision of detection.
Invention content
In view of the deficiencies in the prior art, this patent is intended to provide a kind of kit for being more preferably suitable for unicellular amplification,
On the one hand it is intended to through optimizational primer and amplification step, the unicellular sample after fixing can be efficiently applied to；It simultaneously can by one kind
It is applied in unicellular sample amplification method with the enzymatic treatment step of specific excision uridylate, expansion can be significantly reduced
The error rate of increasing.
In order to achieve the above objects and other related objects, this patent provides a kind of unicellular whole genome amplification reagent
Box, the kit include：
Cell pyrolysis liquid, cell cracking enzyme, correction enzyme, reaction terminating liquid, pre- amplification buffer, whole amplification buffer, magnetic
Pearl；
Further, the cell pyrolysis liquid ingredient includes：50mM KAc, 20mM Tris-Ac, 10mM MgAc, 0.2%
Trition X-100；Can also be 1xPBS, 1xThermo polbuffer, 30mM Tris-HCl, 10mM Tris-HCl；
25mM EDTA；100mM NaCl；0.5%SDS, 10mM Tris-HCl；50mM KCl、10mM Tris-HCl；100mM
EDTA；0.5%SDS, 30mM Tris-HCl；10mM EDTA；1%SDS, 30mM Tris-HCl；2mM KCl, 0.2%
The buffer solutions such as Triton X-100 are most preferably 50mM KAc, 20mM Tris-Ac, 10mM MgAc, 0.2%Trition
X-100。
Further, the cell cracking enzyme includes：QIAGEN protease, 10mg/ml；QIAGEN Proteinase
K, 20mg/ml.
Further, the correction enzyme is the enzyme with glycosylase activity, including but not limited to following one or more of：
UDG enzymes or other DNA glycosylations with this activity can also be used alone in the mixture of UDG enzymes, exonuclease VIII
Enzyme is (including (single-stranded selection uracil-DNA glycosylase, TU mismatched dna glycosylases, the urine with methyl binding domain are phonetic
Pyridine-DNA glycosylases)；
It is to be appreciated that UDG enzymes (also known as urinate phonetic by Uracil-DNA Glycocasylase, uracil-DNA glycosylase
Pyridine-N- glycosylases, UNG) can specific recognition DNA it is single-stranded or double-stranded in uracil residues, and the hydrolysis removal urine from DNA
Pyrimidine residue is the important component during base excision repair.
The existing N- glycosylases activity of E. coli endonuclease VIII (Endonuclease VIII) also has AP- to crack enzyme activity
Property.The pyrimidine bases being damaged on the releasable double-stranded DNA of N- glycosylases activity, generate de- pyrimidine (AP) site.AP- is cracked
Enzymatic activity then respectively in the 3 ' of AP site and 5 ' end cutting phosphodiester bonds, generates 5 ' phosphoric acid and 3 ' phosphate terminals.Two kinds of enzymes can
With effective mistake repaired caused cytosine deamination in amplification and form uracil.
The technical solution of this patent creatively by carrying out enzymatic treatment to DNA after cell lysis procedure, cuts off base
Because of the deoxyuridine acid in group DNA, error rate and the false positive of SNV detections after amplification are reduced.
Further, the reaction terminating liquid includes：Uracil-glycosylase inhibitor；
Further, the pre- amplification buffer includes archaeal dna polymerase, BSA, the second random primer, dNTP DNA polymerizations
Enzyme, seedless sour water；
Further, the pre- amplification buffer is according to following proportioning：
Further, the whole amplification buffer includes archaeal dna polymerase, BSA, the second random primer, dNTP DNA polymerizations
Enzyme, seedless sour water；
Further, the whole amplification buffer is according to following proportioning：
Further, the first random primer structure is：The first primer fixed sequence program-the first primer random sequence-the
One primer, three repetitive sequence, wherein the first primer fixed sequence program such as SEQ ID NO：Shown in 1, specially
ATGACTGATCCTTGTGCTAGAGTGTAC.Wherein the length of the first primer random sequence can be 4~20nt, it is therefore preferable to 4
~10nt, more preferably 6~8nt.
Further, the second random primer structure is：Second the-the second primer of primer fixed sequence program random sequence-the
Two primers, three repetitive sequence, the second primer fixed sequence program such as SEQ ID NO：Shown in 2, specially:
ATGACTGATCCTTGTGCTAGAGTGTAC.Wherein the length of the second primer random sequence can be 4~20nt, it is therefore preferable to 4
~10nt, more preferably 6~8nt.
It is to be appreciated that three repetitive sequences in the first primer are TTT, then three in second primer repeat
Sequence GGG；Three repetitive sequences in the first primer are AAA, then three repetitive sequence GGG in second primer；
Three repetitive sequences in the first primer are TTT, then three repetitive sequence CCC in second primer；Described first
Three repetitive sequences in primer are AAA, then three repetitive sequence CCC in second primer.First random primer
In three repetitive sequences and second random primer in three repetitive sequences can reduce the formation of primer dimer.
Further, the archaeal dna polymerase includes but not limited to following one or more of：Vent archaeal dna polymerases, T7
Archaeal dna polymerase, Bsu archaeal dna polymerases, large fragment, T4 archaeal dna polymerases, DNA polymerase i, (Klenow) large fragment, DNA polymerizations
Enzyme I (E.coli), TherminatorTMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase,
Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst archaeal dna polymerases, overall length, Bst DNA polymerizations
Enzyme, Deep VentRTMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Heat opens
Dynamic Taq archaeal dna polymerases,Thermal starting Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting
Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq archaeal dna polymerases (provide the ThermoPol II without magnesium ion
Buffer solution), Taq archaeal dna polymerases (provideBuffer solution),Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,
Thermal starting surpass fidelity dna polymerase,Super fidelity dna polymerase, KAPA HiFi Uracil+ReadyMix (2x),
The combination of the one or more enzyme such as PfuTurbo Cx thermal starting archaeal dna polymerases.
Further, the magnetic bead includes Agencourt AMPure XP beads or VAHTSTM DNA
Clean Beads、Seq-StarTMDNAClean Beads etc. have the function of the magnetic bead of DNA purifying.It is wherein best to be
Agencourt AMPure XP beads。
It should be noted that using the kit, preferably with the parallel right of a formula 2~5 when sample carries out experimental implementation
According to system, more preferably using 3 times, repetition can be adjusted and supplemented according to experimental result and is tested.
It may be noted that mentioned reagent box is applicable to fixed sample, living cells sample, precious sample, mixing sample, degradation
Sample.
This patent is intended to provide a kind of kit for being more preferably suitable for unicellular amplification, is on the one hand intended to draw by optimization
Object and amplification step can be efficiently applied to the unicellular sample after fixing；It simultaneously can be with specific excision uridine diphosphate by one kind
The enzymatic treatment step of thuja acid is applied in unicellular sample amplification method, can significantly reduce the error rate of amplification.With existing skill
Art is compared, and the kit that this patent is provided has the following advantages：
1. this patent provide kit in sample cracking process by adjusting agent formulations, add in solution cross-linking step,
The modes such as joint magnetic bead PCR optimize, be applicable to it is fixed after unicellular sample amplification, compared in the prior art, greatly
Most amplification methods are only applicable to living cells sample, and poor to passing through the fixed sample amplification efficiency of formaldehyde, strongly limit
Single celled application range.The kit that this patent provides so that expanding object is not only only applicable to living cells sample, also may be used
Suitable for fixed sample, precious sample, mixing sample, degradation sample, use scope is expanded, is had before being more widely applied
Scape.
2. the kit of this patent creatively by it is a kind of can be with the enzymatic treatment step of specific excision uridylate
Applied in unicellular sample amplification method, the error rate of amplification can be significantly reduced.
Description of the drawings
Fig. 1 is the sample false positive SNV ratio charts of a preferred embodiment
Fig. 2 be a preferred embodiment sample S group SNV false positives in different bases mutant proportion figure
Fig. 3 be a preferred embodiment sample US group SNV false positives in different bases mutant proportion figure
Specific embodiment
With reference to specific embodiment, this patent is expanded on further.It should be understood that embodiment solely for the purpose of illustration originally
Patent rather than the range for limiting patent in any way.Test method without specific conditions in the following example, is usually pressed
More solito condition or according to the normal condition proposed by manufacturer.
Unless otherwise defined, it anticipates known to all professional and scientific terms used in text and one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in this patent method.Wen Zhong
The preferred implement methods and materials are for illustrative purposes only.
Use step
Included the following steps using the method that mentioned reagent box carries out unicellular whole genome amplification：
First, sample collection
Unicellular sample after isolated fixation using the 1xPBS of no calcium and magnesium is washed 3 times, is collected in no more than 2 μ l
Not in the 1xPBS of calcium-magnesium-containing, it is placed in 200 μ l PCR thin-wall tubes and saves backup.If cannot be handled in time, in -20 DEG C, -
It is preserved in 80 DEG C or liquid nitrogen.
2nd, cell cracking
1. 10 μ L cell pyrolysis liquids and 0.5 μ L cell cracking enzymes are mixed evenly, for preparing cell cracking mixed liquor.
2. 10 μ L cell pyrolysis liquids (fixed unicellular sample PBS buffer solution weight is added in each unicellular sample
Outstanding, volume is not more than 2 μ L).
It is to be appreciated that the cell pyrolysis liquid ingredient includes but not limited to following one or more：50mM KAc, 20mM
Tris-Ac, 10mM MgAc, 0.2%Trition X-100；Can also be 1xPBS, 1xThermo polbuffer, 30mM
Tris-HCl、10mM Tris-HCl；25mM EDTA；100mM NaCl；0.5%SDS, 10mM Tris-HCl；50mM KCl、
10mM Tris-HCl；100mM EDTA；0.5%SDS, 30mM Tris-HCl；10mM EDTA；1%SDS, 30mM Tris-
HCl；The buffer solutions such as 2mM KCl, 0.2%Triton X-100 are most preferably 50mM KAc, 20mM Tris-Ac, 10mM
MgAc, 0.2%Trition X-100.
3. centrifuging after mixing, it is placed in PCR instrument, 50 DEG C of incubations, 2 hours lytic cells, 80 DEG C are incubated 10min and make egg
White enzyme denaturation.
3rd, enzymatic treatment
1. after the completion of cracking, 1 μ L correction enzymes are added in each sample, is uniformly mixed and is placed in PCR instrument, 37 DEG C of incubations
30min。
It is to be appreciated that the enzyme used in enzymatic treatment in this kit can be the mixed of UDG enzymes and exonuclease VIII
Object is closed, UDG enzymes can also be used alone or other DNA glycosylases with this activity (are urinated phonetic including single-stranded selection
Pyridine-DNA glycosylases, TU mismatched dna glycosylases have the uracil-DNA glycosylase of methyl binding domain).
2. 3 μ L reaction terminating liquids are added in each sample, 37 DEG C of incubation 10min.
4th, solution crosslinking and pre- amplification
1. taking out Agencourt AMPure XP beads from refrigerator, 30min is placed at room temperature for after abundant mixing.
2. adding in 10 μ l Agencourt AMPure XP beads suspensions into above-mentioned lysate, mixing is placed on PCR
On instrument, 65 DEG C of processing 3-6h, best is 3h.
It is to be appreciated that solution cross-linking step in magnetic bead can be Agencourt AMPure XP beads or
VAHTSTM DNA Clean Beads、Seq-StarTMDNAClean Beads etc. have the function of the magnetic bead of DNA purifying.Wherein most
Good is Agencourt AMPure XP beads.
3. terminate to also have 30min from reaction, pre- amplification mixture is configured on ice chest, 4 DEG C spare
Table 1
| Reagent | Volume |
| Pre- amplification buffer | 15 |
| Nuclear free water | Polishing is to 30 μ L |
4. brief centrifugation after cooling, transfers the sample on magnetic frame, discarded supernatant after sample clarification.With 200ul's
The ethyl alcohol of 80% (vol/vol) cleans beads, discards supernatant.
5. it is primary to repeat step 3.
The waiting 6. room temperature is uncapped, until ethyl alcohol fully volatilizees.It adds in the pre- amplification mixtures of 30ul and magnetic bead, mixing is resuspended.
7. said mixture is positioned in PCR instrument, expanded according to the program in following table.
Table 2
Wherein recurring number setting unit, twice of body is unicellular to recommend 6-8 cycle, and monoploid recommends 8-10 cycle, single
Chromosome recommends 10-12 cycle.
5th, amplification eventually
1. 30 μ L ends amplification mixtures (such as following table) are added in each sample.
Table 3
| Reagent | Volume |
| Whole amplification buffer | 15μL |
| Nuclear free water | Polishing is to 30 μ L |
2. mixing is placed in PCR instrument, exponential amplification is carried out according to following condition.
Table 4
Recurring number setting unit in this step, twice of body is unicellular to recommend 16-18 cycle, and monoploid recommends 18-20
A cycle, monosome recommend 20-22 cycle.
6th, magnetic beads for purifying DNA cloning product
Gained DNA product can be used to follow-up a variety of library constructions, such as full extron, full-length genome, target capture etc..
It may be noted that this patent is not only the unicellular full-length genome expansion for providing the application for being very suitable for fixed sample
Increase kit, the kit and its application method may be equally applied to living cells sample, precious sample, mixing sample, degradation sample
This, has expanded sample application range, while reduce error rate, has improved the precision of detection, therefore had a wide range of applications
Prospect.
Embodiment
1. the unicellular leukocyte samples that paraformaldehyde is taken to be captured after fixing by laser micro-cutting method, will detach
To leucocyte be divided into 6 pipes, every 1 cell of pipe.It is divided into two groups of S, US, is respectively designated as S1/S2/S3, US1/US2/US3.Together
Pipe blood takes 500 microlitres of progress whole blood DNA extractings, is named as Sbulk, is compared as follow-up false positive.
2.S groups use MALBAC methods (multiple annealing andlooping-based amplification
Cycles unicellular amplification) is carried out, US group samples are handled according to this kit.Product total amount is as shown in table 5 below after amplification：
Table 5
3. after above-mentioned unicellular amplified production magnetic beads for purifying, the unicellular amplified productions of 300ng is respectively taken to carry out full extron
Library sequencing is built, sequencing depth 100 multiplies, and bulk samples, which are carried out at the same time, builds library sequencing.
4. data analysis, base mutation type between unicellular sample and Bulk samples and ratio and slender are counted
The error rate of born of the same parents' amplification.
5. analysis result is as follows：
1. coverage rate result is as shown in table 6 below：
Table 6
2. using the SNV quantity of the GATK unicellular amplification sequencing datas obtained, as shown in table 7.
Table 7
Analysis result shows, the ratio that the false positive SNV of S groups accounts for total SNV is apparently higher than US groups (0.30%vs0.16%),
Specific sample false positive SNV ratio charts are see Fig. 1.
3. analyze the ratio that different bases mutation is shared in SNV false positives.As a result show that this experiment US group (is split in cell
UDG enzymatic treatments sample is used after solution step) the base mutation quantity of C → T significantly reduces, and whole false positive SNV quantity is also bright
It is aobvious to reduce.S group false positive statistical results are see Fig. 2, and US group false positive statistical results are see Fig. 3.
It can be seen that by above-mentioned data, gained library is in false positive rate statistically generally less than unused UDG enzymatic treatment samples.
False positive rate can effectively be reduced by showing the implementation steps of this patent scheme.This amplification kit can be expanded effectively by fixed list
Cell sample is greatly expanded the application orientation of unicellular amplification；Analysis data result display false positive, which has, to be remarkably decreased,
There is great help to subsequent analysis.
The preferred embodiment of this patent described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour can make many modifications and variations according to the design of this patent.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology according to the design of this patent
Scheme, all should be in the protection domain being defined in the patent claims.
Sequence table
<110>Shanghai Major Biological Medical Technology Co., Ltd.
<120>A kind of unicellular whole genome amplification kit
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atgactgatc cttgtgctag agtgtac 27
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atgactgatc cttgtgctag agtgtac 27
Claims (13)
1. a kind of unicellular whole genome amplification kit, the kit include：Cell pyrolysis liquid, cell cracking enzyme, magnetic bead,
Correct enzyme, reaction terminating liquid, pre- amplification buffer, whole amplification buffer.
A kind of 2. unicellular whole genome amplification kit as described in claim 1, which is characterized in that the cell pyrolysis liquid
It is including but not limited to following any：50mM KAc, 20mM Tris-Ac, 10mM MgAc, 0.2%Trition X-100；
1xPBS、1xThermo polbuffer、30mM Tris-HCl；10mM Tris-HCl、25mM EDTA、100mM NaCl；
0.5%SDS, 10mM Tris-HCl, 50mM KCl；10mM Tris-HCl, 100mM EDTA, 0.5%SDS；30mM Tris-
HCl, 10mM EDTA, 1%SDS；30mM Tris-HCl, 2mM KCl, 0.2%Triton X-100.
3. a kind of unicellular whole genome amplification kit as described in claim 1, which is characterized in that the correction enzyme is tool
There is the enzyme of DNA glycosylases activity.
4. a kind of unicellular whole genome amplification kit as described in claim 1, which is characterized in that the correction enzyme includes
But it is not limited to following one or more：UDG enzymes, exonuclease VIII, single-stranded selection uracil-DNA glycosylase, TU mispairing
DNA glycosylases, the uracil-DNA glycosylase with methyl binding domain.
A kind of 5. unicellular whole genome amplification kit as described in claim 1, which is characterized in that the reaction terminating liquid
Including：Uracil-glycosylase inhibitor.
A kind of 6. unicellular whole genome amplification kit as described in claim 1, which is characterized in that the pre- amplification buffering
Liquid includes archaeal dna polymerase, BSA, dNTP, archaeal dna polymerase, seedless sour water, the first random primer.
7. a kind of unicellular whole genome amplification kit as claimed in claim 6, which is characterized in that described first with power traction
Object structure is：The first primer fixed sequence program-the first primer random sequence-three repetitive sequence of the first primer, wherein described first
Primer fixed sequence program such as SEQ ID NO：Shown in 1, specially ATGACTGATCCTTGTGCTAGAGTGTAC.
8. a kind of unicellular whole genome amplification kit as claimed in claim 7, which is characterized in that the first primer with
Machine sequence length is 4~20nt, it is therefore preferable to 4~10nt, more preferably 6~8nt.
A kind of 9. unicellular whole genome amplification kit as described in claim 1, which is characterized in that the amplification buffering eventually
Liquid includes archaeal dna polymerase, BSA, dNTP, archaeal dna polymerase, seedless sour water, the second random primer.
10. a kind of unicellular whole genome amplification kit as claimed in claim 9, which is characterized in that described second is random
Primer construction is：Second the-the second primer of primer fixed sequence program the-the second primer random sequence, three repetitive sequence, wherein described
Two primer fixed sequence programs such as SEQ ID NO：Shown in 2, have and be:ATGACTGATCCTTGTGCTAGAGTGTAC.
A kind of 11. unicellular whole genome amplification kit as claimed in claim 10, which is characterized in that second primer
Random sequence length is 4~20nt, it is therefore preferable to 4~10nt, more preferably 6~8nt.
12. a kind of unicellular whole genome amplification kit as described in claim 7 or 10, which is characterized in that described first
Three repetitive sequences in primer are TTT, then three repetitive sequence GGG in second primer；In the first primer
Three repetitive sequences are AAA, then three repetitive sequences in second primer
GGG；Three repetitive sequences in the first primer are TTT, then three repetitive sequence CCC in second primer；
Three repetitive sequences in the first primer are AAA, then three repetitive sequence CCC in second primer.
13. a kind of unicellular whole genome amplification kit as described in claim 6 or 9, which is characterized in that the DNA gathers
Synthase includes but not limited to following one or more of：Vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, it is large stretch of
Section, T4 archaeal dna polymerases, DNA polymerase i, (Klenow) large fragment, DNA polymerase i (E.coli), TherminatorTMDNA
Polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, Bst
2.0 archaeal dna polymerases, Bst archaeal dna polymerases, overall length, Bst archaeal dna polymerases, Deep VentRTMArchaeal dna polymerase, VentR
(exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat
Startup Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are big
Segment, Taq archaeal dna polymerases (providing the ThermoPol II buffer solutions without magnesium ion), Taq archaeal dna polymerases (provideBuffer solution),Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting
Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,Super fidelity
Archaeal dna polymerase, KAPA HiFi Uracil+ReadyMix (2x), PfuTurbo Cx thermal starting archaeal dna polymerases.
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Application publication date: 20180615 |