CN105969762A - Reaction reagent used for high-GC-content genome amplification, applications of reaction reagent and kit - Google Patents
Reaction reagent used for high-GC-content genome amplification, applications of reaction reagent and kit Download PDFInfo
- Publication number
- CN105969762A CN105969762A CN201610249847.2A CN201610249847A CN105969762A CN 105969762 A CN105969762 A CN 105969762A CN 201610249847 A CN201610249847 A CN 201610249847A CN 105969762 A CN105969762 A CN 105969762A
- Authority
- CN
- China
- Prior art keywords
- content
- extend
- pcr
- degeneration
- circulations
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Abstract
The invention discloses a reaction reagent used for high-GC-content genome amplification, applications of the reaction reagent and a kit. The reaction reagent used for high-GC-content genome amplification comprises the following components: 20-50 mmol/L of Tris-HCl, 3-5 mmol/L of potassium acetate, 2-4 mmol/L of magnesium acetate, 5-10 V/V% of dimethyl sulfoxide, 30-80 mg/L of bovine serum albumin, 0.003-0.01 V/V% of NP-40, 0.003-0.01 V/V% of Tween20, and 1.0-1.5 mol/L of glycine betaine. The kit comprises primers designed according to target genes and the reaction reagent used for the high-GC-content genome amplification. When the reaction reagent and the kit are used for the high-GC-content genome amplification, the product is high in specificity, and the band is single.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of genome amplification reaction for high GC content
Reagent, the test kit of genome amplification reaction reagent containing described high GC content and anti-for the genome amplification of high GC content
Answer the application of reagent.
Background technology
Polymerase chain reaction (Polymerase Chain Reaction, PCR) is invention in 1985 by K.Mullis
, it is a kind of isothermal DNA amplification.The process that PCR replicates in can simulating DNA body, with original DNA as template, poly-
Under the synergism of synthase and specific primer, dNTPs and buffer system, by degeneration, anneal, extend 3 steps, a few hours
The interior genes of interest fragment of trace being carried out is enriched with amplification, can be amplified to million times under suitable conditions.Greatly facilitate
The mankind obtain minim DNA and study, and improve efficiency.Multiple research field, such as gene clone have been goed deep in PCR application
With expression, DNA sequencing, gene test, genetic modification etc..Along with progress, the popularization of application of round pcr, also it is faced with
New problem.Such as the target gene of some high GC contents, PCR amplification in most cases can not get preferable result, is not
What poor specificity and causing obtained is not genes of interest, it is simply that poor sensitivity causes the amount obtained little.Relative to shape between A, T
Become 2 for hydrogen bond, can be formed three to hydrogen bond between G, C, unwind required can value higher, DNA double chain is relatively difficult to dozen
Open, and template is easily formed stable secondary structure prevention archaeal dna polymerase extension on template DNA chain, thus cause
Expand unsuccessfully.
Studies have reported that some PCR additives can improve the genomic PCR amplification of high GC content in various degree, such as
Glycine betaine, glycerol, DMSO, Methanamide etc., also have some organic solvents, such as 1,2 propylene glycol, ethylene glycol etc..Since 1993,
Etc. Rees.W.A., after reported first glycine betaine can reduce the Tm value of DNA, glycine betaine is just widely used in various PCR,
Act primarily as the effect of two aspects: one is can be with the big minor groove binding in duplex DNA, it is simple to opening of secondary structure, protect
The carrying out of card amplified reaction；Two is as a kind of neutral substance, has protective effect to archaeal dna polymerase, and can reduce PCR expansion
Mass problem in increasing process.DMSO, as a kind of PCR additive, is used for improving optimization PCR condition very early, and its essence is broken
The hydrogen bond structure of bad DNA, promotes the secondary structure of DNA to can be good at opening, and improves the specificity of PCR.Research simultaneously shows
Too high DMSO concentration can affect the activity of archaeal dna polymerase, and then suppression PCR reaction.Organic solvent is the most to varying degrees
Affecting the course of reaction of PCR, tool has some improvement, but in view of organic solvent needs individually to add, due to itself
Viscosity, it may appear that final consumption is not accurate especially, and the most most organic solvents are the most harmful, used
Institute's inconvenience.Therefore, there is suppression PCR reaction, consumption difficulty control in the genomic PCR amplification additive being currently used for improving high GC content
The problem that system is undesirable with susceptiveness with specificity.
Summary of the invention
It is an object of the invention to overcome the above-mentioned deficiency of prior art, it is provided that a kind of genome for high GC content expands
Increase reaction reagent, it is intended to solve the genome amplification reaction reagent being currently used for high GC content and react at suppression PCR, consumption difficulty control
The technical problem that system is undesirable with susceptiveness with specificity.
It is a further object of the present invention to provide test kit and the high GC content of a kind of genome amplification for high GC content
Genome amplification method, it is intended to solve the technical problem that the genomic PCR amplification effect of existing high GC content is undesirable.
In order to realize foregoing invention purpose, as an aspect of of the present present invention, it is provided that a kind of gene for high GC content
Group amplification reaction reagent.The described genome amplification reaction reagent for high GC content contains following component:
Tris-HCl 20~50 mmol/L
Potassium acetate 3 ~ 5 mmol/L
Magnesium acetate 2 ~ 4 mmol/L
Dimethyl sulfoxide 5 ~ 10V/V %
Bovine serum albumin 30 ~ 80 mg/L
NP-40 0.003~0.01V/V %
Tween20 0.003~0.01V/V %
Glycine betaine 1.0 ~ 1.5 mol/L.
As another aspect of the present invention, it is provided that the test kit of a kind of genome amplification for high GC content.Described
Test kit includes the primer according to target gene design, also includes the genome amplification reaction for high GC content of the present invention
Reagent.
As another aspect of the present invention, it is provided that a kind of genome amplification method of high GC content, comprise the steps:
Template DNA is carried out in PCR reaction reagent PCR reaction；Wherein, containing with good grounds target gene in described PCR reaction reagent
The genome amplification reaction reagent for high GC content described in the primer of design and claim 1.
Compared with prior art, the present invention passes through contained for the genome amplification reaction reagent of high GC content the present invention
Synergistic effect between component, has a following Advantageous Effects:
(1) present invention can expand template G/C content for the genome amplification reaction reagent of high GC content and be up to 82%, even more
Height, can be used for the PCR amplification in high GC site in clinical gene detection；
(2) wide accommodation, for commercialization PCR kit different on market, can direct concerted reaction；
(3) can use the unified PCR amplification condition can be to different G/C content, different length size, different primers to carrying out
Amplification；
(4) product specificities for the genome amplification reaction agent amplifies of high GC content is high, band is single, can directly use
In follow-up sequencing reaction and other molecule experiments or Clinical detection.
The present invention is used for the test kit of the genome amplification of high GC content owing to being used for the base of high GC content containing the present invention
Because of group amplification reaction reagent, therefore, it can expand template G/C content and be up to 82%, the highest, can be used for clinical gene detection
The PCR amplification in middle high GC site；Can be for different G/C content, different length size, different primers to using unified PCR to expand
Increasing condition expands；The product specificities of test kit of the present invention amplification is high, band is single, is used directly for follow-up order-checking
Reaction and other molecule experiments or Clinical detection.
The genome amplification method of high GC content of the present invention uses the genome amplification being used for high GC content containing the present invention
The PCR reaction reagent of reaction reagent carries out PCR reaction, it is possible to G/C content is up to 82%, and the most higher amplification template expands
Increase, and make the product specificities height of amplification, band single；And can be for different G/C content, different length size, differences
The unified PCR amplification condition of employing is expanded by primer so that the inventive method technique is relatively easy, improves the effect of amplification
Rate.
Accompanying drawing explanation
Fig. 1 is the pcr amplification product electrophoretogram carried out according to program 1 in case study on implementation 31 of the present invention；Wherein Marker
Size is 2000bp, 1000bp, 800bp, 600bp, 400bp, 200bp;Glue hole is from left to right followed successively by: APOE, PLEC,
rs199726272、PLCB3、Marker、notch3-33F1R1、rs45470261、rs572346021、notch3-33FR、
notch3-10F1R1；
Fig. 2 is the pcr amplification product electrophoretogram carried out according to program 2 in case study on implementation 31 of the present invention；The wherein size of Marker
For 2000bp, 1000bp, 800bp, 600bp, 400bp, 200bp;Glue hole is from left to right followed successively by: APOE, PLEC,
rs199726272、PLCB3、Marker、notch3-33F2R2、rs572346021、notch3-33F1R1、rs45470261、
notch3-10F1R1；
Fig. 3 is corresponding sequence result in case study on implementation 31-33 of the present invention；
Fig. 4 is corresponding sequence result in case study on implementation 31-33 of the present invention.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, right
The present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, not
For limiting the present invention.
Embodiment of the present invention volume parts noted in the disclosure not only may refer to the volume ratio of each component and closes
System, it is also possible to represent each group of concrete volume content, therefore, as long as according to the embodiment of the present invention each component of description compositions
Volume parts scales up or reduces all within embodiment of the present invention description scope of disclosure.Specifically, the present invention is real
Executing the volume parts described in example description can be volume unit known to the biochemistry such as l, L.
The explanation of key word related to the present invention:
Tris-HCl:Tris (hydroxymethyl) aminomethane, three (methylol) aminomethane, molecular formula is
C4H11NO3
DMSO:Dimethyl sulfoxide, dimethyl sulfoxide, a kind of organic compounds containing sulfur, molecular formula is (CH3)2SO
Betaine: glycine betaine
NP-40: lysate (NP-40 Lysis Buffer)
Tween20: polysorbas20
BSA: bovine serum albumin
DNTP:deoxy-ribonucleoside triphosphate, dideoxyribonucleotide triphosphate.
On the one hand, a kind of genome amplification reaction reagent for high GC content is embodiments provided.Described use
Genome amplification reaction reagent in high GC content comprises following component:
Tris-HCl 20~50 mmol/L
Potassium acetate (KOAC) 3 ~ 5 mmol/L
Magnesium acetate (Mg (OAC)2) 2 ~ 4 mmol/L
Dimethyl sulfoxide (DMSO) 5 ~ 10V/V %
Bovine serum albumin (BSA) 30 ~ 80 mg/L
NP-40 0.003~0.01V/V %
Tween20 0.003~0.01V/V %
Glycine betaine (Betaine) 1.0 ~ 1.5 mol/L.
So, the embodiment of the present invention is between the genome amplification reaction reagent of high GC content is by contained component
Synergistic effect so that it can expand template G/C content and be up to 82%, the highest；Can be for different G/C content, different lengths
Size, different primers are to using unified PCR amplification condition amplification, and make amplified production specificity height, band single, permissible
It is directly used in follow-up sequencing reaction and other molecule experiments or Clinical detection.Specifically, certain density DMSO can break
The hydrogen bond of bad DNA, promotes DNA double chain to unwind, and can effectively protect archaeal dna polymerase with the use of certain density glycine betaine
Activity, farthest reduce the reduction of the DNA polymerase activity brought due to DMSO.Thus the effect of the amplification that hits pay dirk
Really.
In one embodiment, the Tris-HCl that this Tris-HCl can select pH to be 9.0.At some specific embodiments
In, can be 20mmol/L, 22mmol/L, 25mmol/L, 26mmol/L, 28mmol/L, 33mmol/L, 36mmol/ at content
L, 38mmol/L, 40mmol/L, 42mmol/L, 44mmol/L, 46mmol/L, 48mmol/L, 50 mmol/L isoconcentrations.
In certain embodiments, the content of potassium acetate can be that 3mmol/L, 4mmol/L, 5 mmol/L etc. are dense
Degree.
In certain embodiments, the content of magnesium acetate can be 2mmol/L, 3mmol/L, 4mmol/L isoconcentration.
In certain embodiments, the content of dimethyl sulfoxide can be 5V/V%, 6V/V %, 7V/V %, 8V/V %,
9V/V %, 10V/V % equal-volume degree.
In certain embodiments, the content of bovine serum albumin can be 30mg/L, 35mg/L, 40mg/L, 45mg/
L, 50mg/L, 55mg/L, 60mg/L, 65mg/L, 70mg/L, 75mg/L, 80mg/L isoconcentration.
In certain embodiments, what the content of NP-40 with Tween20 can be identical differs be 0.003V/V%,
0.004V/V %, 0.005V/V %, 0.006V/V %, 0.007V/V %, 0.008V/V %, 0.009V/V %, 0.01V/V % etc.
Volume percent content.
In certain embodiments, the content of glycine betaine can be 1 mol/L, 1.1mol/L, 1.2mol/L,
1.3mol/L, 1.4mol/L, 1.5mol/L isoconcentration.
By the concentration of above-mentioned each composition is adjusted, it is possible to optimize the synergistic effect between component, thus improve this
The genome of high GC content is expanded by inventive embodiments for the genome amplification reaction reagent of high GC content, and expands product
Thing specificity is high, band is single.
On the other hand, on the basis of above for the genome amplification reaction reagent of high GC content, the embodiment of the present invention
Additionally provide the test kit of a kind of genome amplification for high GC content.Described test kit is except comprising existing PCR institute
Some reagent, it is necessary to include the embodiment of the present invention mentioned above genome amplification reaction reagent for high GC content.
In one embodiment, this genome amplification reaction reagent being used for high GC content can separate guarantor with other reagent in test kit
Deposit and put, time to be amplified, carry out mixed processing in proportionally requirement；Can also be with other reagent in test kit according to necessarily
Ratio mix, formed mix reagent, amplification time directly use.In another embodiment, the embodiment of the present invention is used for
In the test kit of the genome amplification of high GC content, the embodiment of the present invention above of contained amount expands for the genome of high GC content
Increase reaction reagent amount can according to the number of times of amplification and every time consumption required for amplification control flexibly
In a further embodiment, PCR buffer, dNTPs, archaeal dna polymerase are also included contained by embodiment of the present invention test kit
With PCR reagent such as at least one in sterilizing ultra-pure water reagent.Those PCR reagent can also be retained separately placing, time to be amplified
Mixed processing is carried out in proportionally requirement；Can also mix according to certain ratio, form mix reagent, when amplification
Directly use.In a particular embodiment, described archaeal dna polymerase is Taq enzyme.
Owing to the embodiment of the present invention contains the embodiment of the present invention above for the test kit of the genome amplification of high GC content
For the genome amplification reaction reagent of high GC content, therefore, it can expand template G/C content and be up to 82%, the highest, can
The PCR amplification in high GC site in clinical gene detects；Can be for different G/C content, different length size, different primers
Expand using unified PCR amplification condition；The product specificities of test kit of the present invention amplification is high, band is single, permissible
It is directly used in follow-up sequencing reaction and other molecule experiments or Clinical detection.
Another aspect, on the basis of above for the genome amplification reaction reagent of high GC content, the embodiment of the present invention
Additionally provide a kind of genome amplification method of high GC content.This amplification method comprises the steps:
Template DNA is carried out in PCR reaction reagent PCR reaction；Wherein, containing with good grounds target gene in described PCR reaction reagent
The genome amplification reaction reagent for high GC content described in the primer of design and claim 1.
Wherein, template DNA can extract according to target DNA to be amplified temporarily, as used OMEGA company of the U.S.
Test kit D3392-01 extracts from containing the former material of target dna.It addition, this template DNA can be any hope amplification
The DNA of target DNA, especially high GC content.In a particular embodiment, this template DNA can be containing rs545470261；
People's FOXC1 gene of two SNP site of rs572346201, containing exon 33, people's notch3 genetic fragment of exons 10,
Partial sequence in APOE gene containing rs7412, rs429358 SNP site, PLEC gene, the part of PLCB3 genome
Sequence, OBSCN gene etc. containing rs199726272 SNP site.
Owing to being by PCR reaction, therefore, PCR reaction reagent used in embodiment of the present invention method should be understood to
Must include be capable of PCR reaction conventional reagent, as the primer designed according to target gene, PCR buffer, dNTPs,
At least one in archaeal dna polymerase and sterilizing ultra-pure water reagent.Except PCR reaction reagent must include, to be capable of PCR anti-
Outside the conventional reagent answered, in embodiments of the present invention, this PCR reaction reagent must also include the present invention mentioned above in fact
Execute the example genome amplification reaction reagent for high GC content.Owing to this is for the genome amplification reaction reagent of high GC content
Exist, other reagent effects that this genome amplification reaction reagent being used for high GC content reacts with PCR, so that this
Bright embodiment method can be up to 82% to G/C content, and the most higher amplification template expands, and makes the product spy of amplification
The opposite sex is high, band is single；And can be for different G/C content, different length size, different primers to using unified PCR to expand
Increasing condition expands so that the inventive method technique is relatively easy, improves the efficiency of amplification.
In one embodiment, the PCR reaction reagent in embodiment of the present invention method and described template DNA constitute PCR amplification
System, is in terms of 20 volume parts by described PCR amplification system cumulative volume, and described PCR amplification system is as follows:
10 × PCR buffer 2.0 volume parts
10mM dNTPs 0.2 volume parts
Primer 1.0 volume parts in 5 μm ol/L
Primer 1.0 volume parts under 5 μm ol/L
Template DNA 1.0 volume parts
Archaeal dna polymerase 0.2 volume parts
The embodiment of the present invention is for genome amplification reaction reagent 10 volume parts of high GC content
Sterilizing ultra-pure water complements to 20 volume parts.
In further embodiment, the described archaeal dna polymerase contained by PCR amplification system is Taq enzyme.
By controlling the concentration of each component of PCR amplification system such that it is able to optimize the synergistic effect between component, thus carry
The genome of high GC content is expanded by the high embodiment of the present invention for the genome amplification reaction reagent of high GC content, and expands
Increasing product specificities is high, band is single, and can be unified to using for different G/C content, different length size, different primers
PCR amplification condition expand so that the inventive method technique is relatively easy, improve amplification efficiency.
In one embodiment, the response procedures of described PCR reaction is following response procedures one:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s s, 63 ~ 65 DEG C of annealing 30 ~ 45s s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s s, 58 ~ 60 DEG C of annealing 30 ~ 45s s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30 ~ 45s s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 52 DEG C of annealing 30 ~ 45s s, 72 DEG C extend 60,10 circulations；
72 DEG C extend 7min, and 16 DEG C are terminated PCR reaction；
In one embodiment, the response procedures one of above-mentioned PCR reaction is as follows:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 52 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
In another embodiment, the response procedures of above-mentioned PCR reaction is that following response procedures two expands:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 69 ~ 70 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 66 ~ 68 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 63 ~ 65 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 58 ~ 60 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 50 ~ 55 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction.
In one embodiment, the response procedures two of above-mentioned PCR reaction is as follows:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, DEG C annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 68 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction.
PCR amplification system in embodiment of the present invention method contains the genome amplification reaction reagent for high GC content
On the basis of, by the optimal control to amplification program, improve the genome amplification reaction reagent place performance for high GC content
Effect, improves and G/C content is up to 82%, and the most higher amplification template carries out the effect expanded, and the product improving amplification is special
Property high, band is single.
Below in conjunction with specific embodiment to the above-mentioned genome amplification reaction reagent for high GC content, contain for high GC
The test kit of the genome amplification reaction reagent of content and the genome amplification method of high GC content are described in detail.
Genome amplification reaction reagent embodiment for high GC content:
Embodiment 11
Present embodiments provide a kind of genome amplification reaction reagent for high GC content.The described gene for high GC content
Group amplification reaction reagent comprises following component:
Tris-HCl 20~50 mmol/L PH 9.0
Potassium acetate 3 ~ 5 mmol/L
Magnesium acetate 2 ~ 4 mmol/L
Dimethyl sulfoxide 5-10% V/V %
Bovine serum albumin 30 ~ 80 mg/L
NP-40 0.003-0.01% V/V %
Tween20 0.003-0.01%V/V %
Glycine betaine 1.0-1.5 mol/L.
Test kit embodiment for the genome amplification of high GC content:
Embodiment 21
The genome amplification reaction for high GC content increasing above-described embodiment 11 in existing PCR kit respectively tries
Agent, forms the test kit of the genome amplification for high GC content.
The genome amplification method box embodiment of high GC content:
Embodiment 31
The present embodiment 31 provides a kind of rs545470261 expanded in people's FOXC1 gene；Two SNP site of rs572346201
Method.
Prepared by 1.DNA template
Extract the 200 μ l blood DNAs of people with the test kit D3392-01 of OMEGA company of the U.S., obtaining concentration is 55.1ng/ l
DNA, 65 μ l altogether.OD260/280 ratio is 1.94, and the ratio of OD260/230 is 2.1.
2. design of primers
Use rs545470261；Two SNP upstream and downstream each 200bp sequences of rs572346201 are template, set with Oligo7.0 software
Meter primer, product amplification size and G/C content table specific as follows 1 thereof:
Table 1
3.PCR reacts
PCR amplification system see table 2:
Table 2
4.PCR amplification program
Using ABI9700 PCR instrument to carry out PCR reaction, response procedures expands according to the program of following program 1 and program 2 respectively
Increase:
(1) program 1:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 52 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
(2) program 2:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 70 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 68 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
5. result detection
After reaction terminates, taking 5 μ L product, 2% agarose gel electrophoresis detection, electrophoresis result is as shown in Figure 1, 2.Wherein,
Fig. 1 is the amplification electrophoretogram according to program 1.The size of concrete Marker the most in FIG is 2000bp, 1000bp, 800bp,
600bp、400bp、200bp;Glue hole is from left to right followed successively by: APOE, PLEC, rs199726272, PLCB3, Marker,
notch3-33F1R1、rs45470261、rs572346021、notch3-33FR、notch3-10F1R1。
Fig. 2 is the amplification electrophoretogram according to program 2.The size of concrete Marker the most in FIG is 2000bp, 1000bp,
800bp、600bp、400bp、200bp。
Glue hole is from left to right followed successively by: APOE, PLEC, rs199726272, PLCB3, Marker, notch3-33F2R2,
rs572346021、notch3-33F1R1、rs45470261、notch3-10F1R1。
6. sequencing result
Residue product directly send order-checking portion of Aomei tripotassium dicitratobismuthate (Beijing) Gene Tech. Company Limited to check order, and sequencing result site is attached
Part sequence is as shown in Figure 3, Figure 4.
Case study on implementation 32:
The present embodiment provide a kind of expand in people's notch3 gene exon 33, exons 10 Partial Fragment.
Prepared by 1.DNA template
Extract the 200 μ l blood DNAs of people with the test kit D3392-01 of OMEGA company of the U.S., obtaining concentration is 55.1ng/ l
DNA, 65 μ l altogether.OD260/280 ratio is 1.94, and the ratio of OD260/230 is 2.1.
2. design of primers
It is classified as template, with Oligo7.0 software design primer, product amplification size and G/C content thereof by the total order of notch3 gene
Table 3 specific as follows:
Table 3
3.PCR reacts
PCR amplification system see table 4:
Table 4
4.PCR amplification program
Using ABI9700 PCR instrument to carry out PCR reaction, response procedures expands according to the program of following program 1 and program 2 respectively
Increase:
(1) program 1:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 52 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
(2) program 2:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 70 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 68 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
5. result detection
After reaction terminates, take 5 μ L product, 2% agarose gel electrophoresis detection, see Fig. 1,2.
Case study on implementation 33
The present embodiment provides a kind of rs7412, rs429358 SNP site detected in people's APOE gene；Portion in PLEC gene
Rs199726272 SNP site in sub-sequence, the partial sequence of PLCB3 genome, OBSCN gene.
Prepared by 1.DNA template
Extract the 200 μ l blood DNAs of people with the test kit D3392-01 of OMEGA company of the U.S., obtaining concentration is 55.1ng/ l
DNA, 65 μ l altogether.OD260/280 ratio is 1.94, and the ratio of OD260/230 is 2.1.
2. design of primers
According to the gene complete sequence template of the DNA profiling that the present embodiment 1 provides, with Oligo7.0 software design primer, product expands
Increase little and G/C content table specific as follows 5:
Table 5
3.PCR reacts
PCR amplification system see table 6:
Table 6
4.PCR amplification program
Using ABI9700 PCR instrument to carry out PCR reaction, response procedures expands according to the program of following program 1 and program 2 respectively
Increase:
(1) program 1:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 52 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
(2) program 2:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 70 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 68 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 65 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
5, result detection
After reaction terminates, take 5 μ L product, 2% agarose gel electrophoresis detection, see Fig. 1,2.It should be noted that through surveying
Test result shows, the point value chosen in the small size scope of embodiment 11 does following experiment, final gel electrophoresis figure basic respectively
Cause, that is to say that in embodiment 11, the content of small size scope is little on final amplification impact, can obtain high specific product, and
Gel electrophoresis strip is single.
From the test result of above-described embodiment 31-33, the genome amplification method of embodiment of the present invention high GC content
Owing to have employed the genome amplification reaction reagent for high GC content provided containing the embodiment of the present invention, therefore, it is possible to right
The most higher amplification template of G/C content up to 82% expands, and makes the product specificities height of amplification, band single；And
And can make for different G/C content, different length size, different primers to using unified PCR amplification condition to expand
Obtain embodiment of the present invention method technique relatively easy, improve the efficiency of amplification.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Any amendment, equivalent and the improvement etc. made within principle, should be included within the scope of the present invention.
Claims (8)
1. the genome amplification reaction reagent for high GC content, it is characterised in that comprise following component:
Tris-HCl 20~50 mmol/L
Potassium acetate 3 ~ 5 mmol/L
Magnesium acetate 2 ~ 4 mmol/L
Dimethyl sulfoxide 5 ~ 10V/V %
Bovine serum albumin 30 ~ 80 mg/L
NP-40 0.003~0.01V/V %
Tween20 0.003~0.01V/V %
Glycine betaine 1.0 ~ 1.5 mol/L.
2. the test kit for the genome amplification of high GC content, it is characterised in that include drawing according to target gene design
Thing, also includes the genome amplification reaction reagent for high GC content described in claim 1.
Test kit the most according to claim 2, it is characterised in that: also include PCR buffer, dNTPs, archaeal dna polymerase and
At least one in sterilizing ultra-pure water reagent.
Test kit the most according to claim 3, it is characterised in that: described archaeal dna polymerase is Taq enzyme.
5. a genome amplification method for high GC content, comprises the steps:
Template DNA is carried out in PCR reaction reagent PCR reaction；Wherein, containing with good grounds target gene in described PCR reaction reagent
The genome amplification reaction reagent for high GC content described in the primer of design and claim 1.
Test kit the most according to claim 5, it is characterised in that: the response procedures of described PCR reaction is following reaction interval
Sequence one or response procedures two:
Response procedures one:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 63 ~ 65 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 58 ~ 60 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 55 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 52 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction；
Response procedures two:
95 DEG C of thermal starting 5min, 1 circulation；
94 DEG C of degeneration 30s, 69 ~ 70 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 66 ~ 68 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 5 circulations；
94 DEG C of degeneration 30s, 63 ~ 65 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 58 ~ 60 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 10 circulations；
94 DEG C of degeneration 30s, 50 ~ 55 DEG C of annealing 30 ~ 45s, 72 DEG C extend 60s, 9 circulations；
72 DEG C extend 7 min, and 16 DEG C are terminated PCR reaction.
7. according to the test kit described in claim 5 or 6, it is characterised in that: described PCR reaction reagent also includes 10 × PCR
Buffer, dNTPs, archaeal dna polymerase and sterilizing ultra-pure water, described PCR reaction reagent and described template DNA constitute PCR and expand body
System, is in terms of 20 volume parts by described PCR amplification system cumulative volume, and described PCR amplification system is as follows:
10 × PCR buffer 2.0 volume parts
10mM dNTPs 0.2 volume parts
Primer 1.0 volume parts in 5 μm ol/L
Primer 1.0 volume parts under 5 μm ol/L
Template DNA 1.0 volume parts
Archaeal dna polymerase 0.2 volume parts
Described genome amplification reaction reagent 10 volume parts for high GC content
Sterilizing ultra-pure water complements to 20 volume parts.
Test kit the most according to claim 7, it is characterised in that: described archaeal dna polymerase is Taq enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249847.2A CN105969762A (en) | 2016-04-21 | 2016-04-21 | Reaction reagent used for high-GC-content genome amplification, applications of reaction reagent and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249847.2A CN105969762A (en) | 2016-04-21 | 2016-04-21 | Reaction reagent used for high-GC-content genome amplification, applications of reaction reagent and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105969762A true CN105969762A (en) | 2016-09-28 |
Family
ID=56993790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610249847.2A Pending CN105969762A (en) | 2016-04-21 | 2016-04-21 | Reaction reagent used for high-GC-content genome amplification, applications of reaction reagent and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105969762A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434924A (en) * | 2016-09-29 | 2017-02-22 | 北京全式金生物技术有限公司 | Kit and method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction) |
| CN111304285A (en) * | 2019-12-13 | 2020-06-19 | 北京先声医学检验实验室有限公司 | Urinary metagenome sample library building and detecting method based on nanopore sequencing platform |
| CN112831547A (en) * | 2021-01-13 | 2021-05-25 | 南京诺唯赞生物科技股份有限公司 | qPCR additive for high GC fragment amplification and amplification method |
| CN116904583A (en) * | 2023-09-08 | 2023-10-20 | 北京贝瑞和康生物技术有限公司 | Detection probe set, kit and method for dynamic mutation of STR and VNTR gene loci |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643762A (en) * | 2009-09-02 | 2010-02-10 | 陈依军 | PCR amplification system and PCR amplification method for high GC content gene |
| EP2050819B1 (en) * | 2006-07-26 | 2012-03-28 | Nishikawa Rubber Co., Ltd. | Method for amplification of nucleotide sequence |
| CN102465120A (en) * | 2010-11-10 | 2012-05-23 | 深圳华大基因科技有限公司 | Fluorescence quantitative PCR reaction solution and fluorescence quantitative PCR method |
| CN103562407A (en) * | 2011-02-09 | 2014-02-05 | 伯乐生命医学产品有限公司 | Analysis of nucleic acids |
| CN103981172A (en) * | 2014-03-27 | 2014-08-13 | 江苏佰龄全基因生物医学技术有限公司 | PCR amplification additive composition used for high-GC gene, and PCR amplification method of high-GC gene |
| WO2015048571A3 (en) * | 2013-09-26 | 2015-07-02 | Bio-Rad Laboratories, Inc. | Methods and compositions for chromosome mapping |
-
2016
- 2016-04-21 CN CN201610249847.2A patent/CN105969762A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2050819B1 (en) * | 2006-07-26 | 2012-03-28 | Nishikawa Rubber Co., Ltd. | Method for amplification of nucleotide sequence |
| CN101643762A (en) * | 2009-09-02 | 2010-02-10 | 陈依军 | PCR amplification system and PCR amplification method for high GC content gene |
| CN102465120A (en) * | 2010-11-10 | 2012-05-23 | 深圳华大基因科技有限公司 | Fluorescence quantitative PCR reaction solution and fluorescence quantitative PCR method |
| CN103562407A (en) * | 2011-02-09 | 2014-02-05 | 伯乐生命医学产品有限公司 | Analysis of nucleic acids |
| WO2015048571A3 (en) * | 2013-09-26 | 2015-07-02 | Bio-Rad Laboratories, Inc. | Methods and compositions for chromosome mapping |
| CN103981172A (en) * | 2014-03-27 | 2014-08-13 | 江苏佰龄全基因生物医学技术有限公司 | PCR amplification additive composition used for high-GC gene, and PCR amplification method of high-GC gene |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434924A (en) * | 2016-09-29 | 2017-02-22 | 北京全式金生物技术有限公司 | Kit and method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction) |
| CN111304285A (en) * | 2019-12-13 | 2020-06-19 | 北京先声医学检验实验室有限公司 | Urinary metagenome sample library building and detecting method based on nanopore sequencing platform |
| CN111304285B (en) * | 2019-12-13 | 2020-09-29 | 北京先声医学检验实验室有限公司 | Urinary metagenome sample library building and detecting method based on nanopore sequencing platform |
| CN112831547A (en) * | 2021-01-13 | 2021-05-25 | 南京诺唯赞生物科技股份有限公司 | qPCR additive for high GC fragment amplification and amplification method |
| CN116904583A (en) * | 2023-09-08 | 2023-10-20 | 北京贝瑞和康生物技术有限公司 | Detection probe set, kit and method for dynamic mutation of STR and VNTR gene loci |
| CN116904583B (en) * | 2023-09-08 | 2024-02-02 | 北京贝瑞和康生物技术有限公司 | Detection probe set, kit and method for dynamic mutation of STR and VNTR gene loci |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105969762A (en) | Reaction reagent used for high-GC-content genome amplification, applications of reaction reagent and kit | |
| Morgante et al. | Transposable elements and the plant pan-genomes | |
| WO2010141390A3 (en) | Nucleotide transient binding for sequencing methods | |
| DE602007013223D1 (en) | CHEMICALLY MODIFIED OLIGONUCLEOTIDE PRIMERS FOR NUCLEIC ACID AMPLIFICATION | |
| WO2011055916A3 (en) | Method for detecting the methylation of colon-cancer-specific methylation marker genes for colon cancer diagnosis | |
| WO2009092035A3 (en) | Methods and compositions for the analysis of biological molecules | |
| JP2011526788A5 (en) | ||
| MX2007009809A (en) | Selection probe amplification. | |
| PT1907588E (en) | Assays for resistance to echinocandin-class drugs | |
| US20210010064A1 (en) | Enrichment of nucleic acids | |
| EP3620536A1 (en) | Method for predicting sex in fish | |
| WO2007002031A3 (en) | Dna polymerase blends and mutant dna polymerases | |
| Taniguchi et al. | Discrimination Between 8‐Oxo‐2′‐Deoxyguanosine and 2′‐Deoxyguanosine in DNA by the Single Nucleotide Primer Extension Reaction with Adap Triphosphate | |
| Azevedo et al. | Colony PCR | |
| DE602008003757D1 (en) | SELECTION AND ENRICHMENT OF PROTEINS BY IN VITRO LIBRARY | |
| CN108192955A (en) | A kind of low frequency mutated DNA fragment detection method and library method for building up | |
| Neo et al. | Visual SNP genotyping using asymmetric PCR and split DNA enzymes | |
| CN112105746B (en) | RNA chemical modification single-gene single-base resolution detection method | |
| CN101392282B (en) | Mixed solution special for PCR for amplifying high GC content fragment and use thereof | |
| CN101381769B (en) | Molecular identification method for siniperca kneri garman and siniperca scherzeri and kit | |
| CN103757094B (en) | A kind of method based on fluorescent PCR detection TYMS gene pleiomorphism | |
| ES2875758T3 (en) | Set of primers and method for the detection and identification of mussel species of the genus mytilus | |
| RU2016126512A (en) | METHOD FOR DETERMINING A MUTANT GENE BY POLYMERASE CHAIN REACTION IN REAL TIME USING DNA POLYMERASE WITH INHIBITED 5'-FLEP-ENDONUCLEASE ACTIVITY | |
| WO2016207857A1 (en) | Set of primers and method for detecting and identifying mussel species of the genus mytilus, using high-resolution melting and pcr | |
| CN100584958C (en) | Method for obtaining molecular marker for identifying three line hybrid rice maintainer line character |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160928 |
|
| RJ01 | Rejection of invention patent application after publication |