CN109852612A - A kind of hair clip Mdification primer of combination universal fluorescent primer - Google Patents
A kind of hair clip Mdification primer of combination universal fluorescent primer Download PDFInfo
- Publication number
- CN109852612A CN109852612A CN201910141427.6A CN201910141427A CN109852612A CN 109852612 A CN109852612 A CN 109852612A CN 201910141427 A CN201910141427 A CN 201910141427A CN 109852612 A CN109852612 A CN 109852612A
- Authority
- CN
- China
- Prior art keywords
- primer
- hair clip
- mdification
- sequence
- universal fluorescent
- 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
Abstract
The present invention relates to a kind of hair clip Mdification primer of combination universal fluorescent primer, kit and the applications in single nucleotide polymorphism detection.The hair clip Mdification primer includes into hairpin, universal fluorescent primer binding sequence and distinguished sequence.Hair clip Mdification primer provided by the invention is cheap and specific height, substantially reduces cost with general fluorescent primer, other required experiment consumptive materials are also cheap and easy to get；Capillary Electrophoresis is carried out after PCR, can intuitively see genotype results, shortens detection cycle, improves detection efficiency；The present invention uses Universal fluorescence probe, only needs to design clamp primers for different loci, substantially reduces research and development cost.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of hair clip Mdification primer of combination universal fluorescent primer, examination
Agent box and the application in single nucleotide polymorphism detection.
Background technique
As the third generation molecular labeling for most having development potentiality in recent years, single nucleotide polymorphism (single
Nucleotide polymorphism, SNP) it is widely applied in genetic analysis.It is in human heritable mutation
One of the most common type accounts for 80% or more of all known polymorphisms.SNP is a kind of label of two condition, by the conversion of single base
It, can also be caused by the insertion of base or missing or caused by transversion.SNP both may be in gene order, it is also possible to gene with
On outer non-coding sequence.
There are many polymorphisms of technology detection mononucleotide, including generation sequencing, the sequencing of two generations, genetic chip at present
Technology, ApoE gene technology (AS-PCR), denaturing high-performance chromatography (DHPLC), mass spectrum detection, high score
Resolution solubility curve etc..Wherein the advantage of ApoE gene technology (AS-PCR) is especially prominent in specificity, but
Though finding that the primer specificity designed according to AS-PCR is good in experiment in recent years, but still there is non-specific amplification, even if
Mispairing is introduced in primer, is still reached to less than ideal effect.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of hair clip Mdification primer of combination universal fluorescent primer, with gram
Existing AS-PCR technology terminal bases fallibility is taken with resolution ratio is not high, is also easy to produce the shortcomings that non-specific amplification.
The present invention provides a kind of hair clip Mdification primers of combination universal fluorescent primer, including at hairpin, general glimmering
Light primer binding sequence and distinguished sequence, it is described to be matched at hairpin and 3 ' end part base sequence complementary of primer but be mutated alkali
Exposed not complementary pairing at base, the universal fluorescent primer binding sequence is identical as the upstream primer sequence of universal fluorescent primer,
The distinguished sequence is used in conjunction with template.
Further, described to be located at the end of hair clip Mdification primer 5 ' at hairpin, length is 9~15 bases, the primer
Itself forms hairpin structure in its natural state.It is described to hold exposed single base only mutual with special base at hairpin 3 '
It recruits pair, primer mispairing probability substantially reduces, and further more accurately distinguishes different mutational sites.
Further, the genotype different for same SNP, a kind of universal fluorescent primer binding sequence ratio of genotype are another
A kind of long 2~4 bases of the universal fluorescent primer binding sequence of genotype, and grown in 3 ' the end sides in this region.It is general glimmering
Light primer complementary with the product of universal fluorescent primer binding sequence can combine, and expanded, make product with fluorescence signal, energy
Enough carry out Capillary Electrophoresis.
Further, the distinguished sequence is located at the end of hair clip Mdification primer 3 ', and length is 10~18 bases.
Further, the genotype different for same SNP, a kind of distinguished sequence of genotype and another genotype
The last one base of distinguished sequence is different, and the base mismatch of introducing is different.
Further, the corresponding downstream primer of the hair clip Mdification primer by universal fluorescent primer downstream sequence binding region and
Downstream primer is successively spliced.In PCR, the corresponding two kinds of clamp primers of each SNP and a kind of downstream primer, at this point, hair
It presss from both sides primer and forms competitive relation, advantageously form higher specificity.
Further, the base that the upstream primer 5 ' of universal fluorescent primer is held is marked with fluorophor.The fluorescent base
Group includes FAM, VIC, HEX, ROX, TaxasRed or CY5, preferably FAM, HEX.
In the present invention, a set of universal fluorescent primer can be used for all genotype, using the principle of nest-type PRC,
In preceding several circulations, expanded with clamp primers, make product with can with the sequence in conjunction with universal fluorescent primer, in conjunction with
Afterwards, it is expanded, adds fluorescence signal for target area PCR product, allow to pass through detection on Capillary Electrophoresis platform
Fluorescence signal judges different genotype.
The present invention is when carrying out multiplex PCR, and when different loci product segment difference, multidigit point, which only needs to share a set of fluorescence, to be drawn
Object can realize the detection in multiple sites, greatly reduce cost, simplify experimental implementation, be easier to promote and use.
The present invention also provides a kind of kits comprising hair clip Mdification primer.Kit further includes dNTPs, Taq DNA
Polymerase, Mg2+, at least one of PCR reaction buffer.
The present invention also provides a kind of application of hair clip Mdification primer in single nucleotide polymorphism detection.
Specifically, hair clip Mdification primer of the invention, universal fluorescent primer are matched architectonical, PCR amplification in kit
After carry out Capillary Electrophoresis, two kinds of genotype are the universal fluorescent primer binding zone T primer ratio C primers according to two kinds of clamp primers
What long 3 bases were completed, in Capillary Electrophoresis genotype can be judged according to the presence or absence of different location peak.
Fig. 1 be same SNP the corresponding primer of different genotype, the two the difference is that: 1) region 3-1 and 3-2 is removed
Different outer, the also artificial introducing mispairing of the last one base；2) 1-1 and the region 1-2 be artificially introduced it is different at mutation；3)2-
2~4 bases more than 2-2 ratio 2-1 in 1 and 2-2.
Fig. 2 be clamp primers in conjunction with template when schematic diagram.Wherein A is the state that upstream clamp primers trail, when
When 3 ' ends are with template matching, then it can extend；When mismatch, it can be split away off from template, form hairpin structure again.In head
It is that leading different genotype special primer 3 ' holds exposed base specific recognition template DNA with hair clip special primer in wheel circulation,
Hairpin structure is opened, to form the product compared with long segment, the fragment products once being formed, the universal fluorescent primer of high concentration into
And identify the universal sequence in long segment, the product compared with short-movie section is amplified, while making different genotype Product Labeling phases
The fluorescence answered, and then the fluorescence signal of different location can be collected on the platforms such as ABI 3730, distinguish the gene of different samples
Kenel.
Beneficial effect
Hair clip Mdification primer provided by the invention is cheap and specific height, substantially reduces with general fluorescent primer
Cost, other required experiment consumptive materials are also cheap and easy to get；Capillary Electrophoresis is carried out after PCR of the present invention, it can be intuitive
See genotype results, shorten detection cycle, improves detection efficiency；The present invention uses Universal fluorescence probe, for different positions
Point only needs to design clamp primers, substantially reduces research and development cost, simplifies experimental implementation, is easier to promote and use.
Detailed description of the invention
Fig. 1 is the corresponding primer of different genotype of same SNP；Wherein, 1-1 and 1-2 is into hairpin, 2-1 and 2-2
Universal fluorescent primer binding sequence, 3-1 and 3-2 are distinguished sequences, X and Y the last one base of primer thus, can with it is complementary
The sample of pairing is combined and is expanded.
Fig. 2 be hair clip Mdification primer of the present invention in conjunction with template when schematic diagram；Wherein, A is the stretching, extension of upstream clamp primers
The state opened (when 3 ' ends are with template matching, then can extend；It when mismatch, can be split away off from template, form hair again
Clamping structure), 4 be universal fluorescent upstream primer, and identical as the sequence of universal fluorescent primer binding sequence of clamp primers, 5 and 6 are
Downstream primer corresponding to clamp primers, 5 sequence is identical as the sequence of universal fluorescent downstream primer 7,6 sequence and template energy
Enough complementations.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Test method without specific conditions in embodiment, usually according to normal condition, such as molecular cloning protocols handbook,
Or according to condition proposed by reagent manufacturer.All inorganic chemical reagents and organic solvent are limited purchased from Solution on Chemical Reagents in Shanghai
Company, dNTPs are purchased from Dalian treasured biotech firm, and Taq archaeal dna polymerase is purchased from Fei Peng Biological Co., Ltd., and primer is by general
The synthesis of biosystem (Anhui) Co., Ltd, people's blood DNA extracting, which uses, likes biotechnology (Hangzhou) Co., Ltd (Axygen that pursues progress
Company) blood genome Miniprep Kit.
Embodiment 1
AS-PCR, which is carried out, using clamp primers detects single nucleotide polymorphism (single nucleotide
Polymorphism, SNP).
Sequence is searched and design of primers: according to the snp database of NCBI, finding out in human genome and mankind's folic acid
The target zone sequence in relevant 3 sites of metabolic capability carries out design of primers according to general rule using Oligo 6.0, and
Upstream universal sequence and downstream universal sequence are manually added respectively at the end of primer upstream and downstream 5 ' of design, are then chemically closed
At upstream and downstream special primer (table 1).
1 SNP of table detects special primer
Note: HP represents clamp primers, and wherein the capitalization of first part is into hairpin, the small letter of second part
Mother is universal fluorescent primer binding sequence, and Part III is distinguished sequence, the lowercase of the lowest be artificially introduced it is prominent
Become.
DNA extracting: human blood cell uses blood genome Miniprep Kit, is stripped to obtain DNA to specifications,
DNA mass and concentration are determined with 0.8% agarose gel electrophoresis.
1. unit point base detects AS-PCR
PCR, which chooses, chooses No. 4 primers (for detecting the site rs1801131 A allele specific upstream primer), 5 in table 1
(for detecting the site rs1801131 C allele specific upstream primer, than No. 1 primer is more 3 on universal sequence for number primer
Base, convenient for distinguishing), the mixing of No. 6 primers (for detecting the special downstream primer of the site rs1801131 C and G allele) it is special
Different amplimer mix, chooses 10, No. 11 primers and is mixed into fluorescent universal primer, carries out PCR reaction to single sample.PCR amplification
System specifically: the 15ng DNA of each sample is added separately in respective 15 μ L Taq enzyme system, wherein Taq enzyme system packet
Containing each primer of 0.002~0.2uM, 1.5ul dNTPs (2.5mM), 1.5ul solution (10x), 3ul HSHiTaq
Buffer(Mg2+Plus), 1U Taq enzyme, and covered using mineral oil；Negative control, response procedures are set simultaneously are as follows: 95 DEG C of denaturation
10min；98 DEG C of denaturation 10s, 57 DEG C of renaturation 2min, 68 DEG C of extension 2min, 4 recycle；98 DEG C of denaturation 10s, 57 DEG C of renaturation 45s,
68 DEG C of extension 2min, 10 circulations；98 DEG C of denaturation 10s, 65 DEG C of renaturation 45s, 68 DEG C of extension 2min, 35 recycle；68 DEG C,
30min。
After amplification, gained PCR product is analyzed on 3730 sequenator of ABI, passes through corresponding two kinds of equipotential bases
Because of the peak height ratio of fluorescence, parting is carried out, parting formula is FA=H1/ (H1+H2), FC=H2/ (H1+H2), wherein H1 is indicated
A kind of genotype (A type is indicated in example), H2 indicate another genotype (c-type is indicated in example).FAWhen value is 1, AA is indicated
It is homozygous；FCWhen value is 1, indicate that CC is homozygous, FAFCHave numerical value and it is close when, indicate AC heterozygous.
It is experimentally confirmed that hairpin structure primer AS-PCR genotyping result is consistent with actual conditions (table 2).
2 site rs1801131 hairpin structure primer AS-PCR result data of table
2. multidigit point base detects AS-PCR
1~9 primer is mixed into primer mix in selection table in PCR selection table 1, chooses 10, No. 11 primers and is mixed into fluorescence
Universal primer carries out multi-PRC reaction to single sample.PCR amplification system specifically: the 15ng DNA of each sample adds respectively
Enter into respective 15 μ L Taq enzyme system, wherein Taq enzyme system includes each primer of 0.002~0.2uM, 1.5ul dNTPs
(2.5mM), 1.5ul solution (10x), 3ul HSHiTaq Buffer (Mg2+Plus), 1U Taq enzyme, and use mineral oil
Covering；Negative control, response procedures are set simultaneously are as follows: 95 DEG C of denaturation 10min；98 DEG C of denaturation 10s, 57 DEG C of renaturation 2mins, 68 DEG C are prolonged
Stretch 2min, 4 circulations；98 DEG C of denaturation 10s, 57 DEG C of renaturation 45s, 68 DEG C of extension 2min, 10 recycle；98 DEG C of denaturation 10s, 65 DEG C
Renaturation 45s, 68 DEG C of extension 2min, 35 recycle；68 DEG C, 30min.
After amplification, gained PCR product is analyzed on 3730 sequenator of ABI, passes through phase for each site
The peak height ratio for answering two kinds of allele fluorescence, judges genotype, the results are shown in Table 3.
3 multidigit point hairpin structure primer AS-PCR result data of table
SEQUENCE LISTING
<110>the Wuxi wing and Applied Biotechnology Co., Ltd
Donghua University
<120>a kind of hair clip Mdification primer of combination universal fluorescent primer
<130> 1
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 54
<212> DNA
<213>artificial sequence
<400> 1
caatttcatc atcgagtgca ctgctgtcgt gtggaggcgt gatgatgaaa ttgg 54
<210> 2
<211> 58
<212> DNA
<213>artificial sequence
<400> 2
ctatttcatc atcagagtgc actgctgtcg tgtggagtag gcgtgatgat gaaataga 58
<210> 3
<211> 39
<212> DNA
<213>artificial sequence
<400> 3
gtgtcctgac gagtgctcac cagagctttg aggctgacc 39
<210> 4
<211> 54
<212> DNA
<213>artificial sequence
<400> 4
cgtcactggt cacgagtgca ctgctgtcgt gtggagggag ctgaccagtg acga 54
<210> 5
<211> 59
<212> DNA
<213>artificial sequence
<400> 5
ctttactggt cagggagtgc actgctgtcg tgtggagtag cggagctgac cagtaaagc 59
<210> 6
<211> 38
<212> DNA
<213>artificial sequence
<400> 6
gtgtcctgac gagtgctcac caggtgacca ttccggtt 38
<210> 7
<211> 54
<212> DNA
<213>artificial sequence
<400> 7
ggtgagcaag cgagtgcact gctgtcgtgt ggagatacca cagcttgctc acct 54
<210> 8
<211> 56
<212> DNA
<213>artificial sequence
<400> 8
cgtgagcaag cgagtgcact gctgtcgtgt ggagtagatc cacagcttgc tcacgc 56
<210> 9
<211> 38
<212> DNA
<213>artificial sequence
<400> 9
gtgtcctgac gagtgctcac cagtgctaca cagcaggg 38
<210> 10
<211> 23
<212> DNA
<213>artificial sequence
<400> 10
gagtgcactg ctgtcgtgtg gag 23
<210> 11
<211> 23
<212> DNA
<213>artificial sequence
<400> 11
gtgtcctgac gagtgctcac cag 23
Claims (10)
1. a kind of hair clip Mdification primer of combination universal fluorescent primer, from 5 ' to 3 ' successively include into hairpin, universal fluorescent
Primer binding sequence and distinguished sequence, it is described to form hair clip pairing at hairpin and 3 ' end part base sequence of primer but be mutated
The amplified production of the exposed not complementary pairing of base at site, the universal fluorescent primer binding sequence is used for and universal fluorescent primer
In conjunction with and carry out the amplification of universal fluorescent primer, the distinguished sequence is used in conjunction with template.
2. hair clip Mdification primer according to claim 1, it is characterised in that: for different genotype, described is general
3 ' end regions of fluorescent primer binding sequence have the difference in length of 2~4 bases.
3. hair clip Mdification primer according to claim 1, it is characterised in that: for different genotype, described hair clip
Hold the last one base different in the 3 ' of Mdification primer.
4. hair clip Mdification primer according to claim 1, it is characterised in that: for different genotype, in described hair
3 ' the ends for pressing from both sides Mdification primer introduce base mismatch.
5. hair clip Mdification primer according to claim 1, it is characterised in that: it is described at hairpin length be 9~15
Base；The distinguished sequence length is 10~18 bases.
6. hair clip Mdification primer according to claim 1, it is characterised in that: draw in the corresponding downstream of the hair clip Mdification primer
Object is made of the downstream sequence combined area of described universal fluorescent primer and downstream distinguished sequence.
7. hair clip Mdification primer according to claim 1, it is characterised in that: 5 ' ends of described universal fluorescent primer have
Fluorescence marker groups.
8. hair clip Mdification primer according to claim 1, it is characterised in that: described hair clip Mdification primer such as SEQ ID
Shown in NO.1,2,4,5,7,8.
9. hair clip Mdification primer according to claim 1, it is characterised in that: described universal fluorescent primer such as SEQ ID
Shown in NO.10.
10. a kind of kit comprising hair clip Mdification primer described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910141427.6A CN109852612A (en) | 2019-02-26 | 2019-02-26 | A kind of hair clip Mdification primer of combination universal fluorescent primer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910141427.6A CN109852612A (en) | 2019-02-26 | 2019-02-26 | A kind of hair clip Mdification primer of combination universal fluorescent primer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109852612A true CN109852612A (en) | 2019-06-07 |
Family
ID=66899045
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910141427.6A Pending CN109852612A (en) | 2019-02-26 | 2019-02-26 | A kind of hair clip Mdification primer of combination universal fluorescent primer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109852612A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100330574A1 (en) * | 2009-06-29 | 2010-12-30 | Whitman Douglas F | Chimeric primers with hairpin conformations and methods of using same |
| CN105624296A (en) * | 2016-01-28 | 2016-06-01 | 屈强 | Universal fluorescent hairpin primers for detecting gene polymorphism on basis of ARMS (amplification refractory mutation system) fluorescent PCR (polymerase chain reaction) process |
| CN106282353A (en) * | 2016-08-26 | 2017-01-04 | 上海翼和应用生物技术有限公司 | A kind of method utilizing clamp primers to carry out multiplex PCR |
| CN107937493A (en) * | 2017-12-06 | 2018-04-20 | 上海翼和应用生物技术有限公司 | A kind of hair clip Mdification primer for allele PCR |
| US20180274029A1 (en) * | 2017-03-24 | 2018-09-27 | Bio-Rad Laboratories, Inc. | Universal hairpin primers |
-
2019
- 2019-02-26 CN CN201910141427.6A patent/CN109852612A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100330574A1 (en) * | 2009-06-29 | 2010-12-30 | Whitman Douglas F | Chimeric primers with hairpin conformations and methods of using same |
| CN105624296A (en) * | 2016-01-28 | 2016-06-01 | 屈强 | Universal fluorescent hairpin primers for detecting gene polymorphism on basis of ARMS (amplification refractory mutation system) fluorescent PCR (polymerase chain reaction) process |
| CN106282353A (en) * | 2016-08-26 | 2017-01-04 | 上海翼和应用生物技术有限公司 | A kind of method utilizing clamp primers to carry out multiplex PCR |
| US20180274029A1 (en) * | 2017-03-24 | 2018-09-27 | Bio-Rad Laboratories, Inc. | Universal hairpin primers |
| CN107937493A (en) * | 2017-12-06 | 2018-04-20 | 上海翼和应用生物技术有限公司 | A kind of hair clip Mdification primer for allele PCR |
Non-Patent Citations (1)
| Title |
|---|
| 王瑞恒等: "基于等位基因特异性PCR原理建立的SNP分型新方法", 《法医学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1896284B (en) | Method for identifying allelic gene type | |
| CN103045591B (en) | HLA gene specific PCR amplification primer, HLA typing method and kit | |
| CN107385064B (en) | Fluorescence labeling composite amplification kit for simultaneously amplifying human autosomal SNP and STR loci and application thereof | |
| CN101532051B (en) | Method for detecting the polymorphism of ADH2 genes | |
| CN105755109B (en) | A kind of new phenylketonuria gene screening and diagnosis system and kit | |
| WO2012037882A1 (en) | Dna tags and use thereof | |
| WO2011023131A1 (en) | Composite amplification kits of 20 short tandem repeats | |
| CN110951828A (en) | Design method of primer and probe | |
| CN107354211B (en) | Forest musk deer four-base microsatellite genetic marker locus and screening method thereof | |
| CN110578009A (en) | Multiplex amplification detection kit containing 40 InDel genetic polymorphic sites of human X chromosome | |
| JP2020536525A (en) | A method for concentrating the probe and the target region to which it is applied for high-throughput sequencing | |
| CN108103164A (en) | A kind of method that copy number variation is detected using multi-fluorescence competitive PCR | |
| CN104178566A (en) | Multiplex fluorescence PCR (polymerase chain reaction) universal adapter for microsatellite detection, and detection method and application thereof | |
| CN109295500B (en) | Single cell methylation sequencing technology and application thereof | |
| CN107988385B (en) | Method for detecting marker of PLAG1 gene Indel of beef cattle and special kit thereof | |
| WO2017185758A1 (en) | Primer, probe, kit, and method for microchimerism assay and individual recognition | |
| CN107937493B (en) | Hairpin modified primer for allele PCR | |
| CN101985659A (en) | Kit for testing schizophrenia related gene and preparation method thereof | |
| CN108410972B (en) | Genotyping detection kit for 23 genetic loci of human Rh blood group | |
| CN108359713B (en) | Screening method of gene polymorphism detection probe | |
| CN107988334B (en) | Method for SNP typing by direct PCR of oral swab | |
| CN110628920A (en) | Fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof | |
| CN109852612A (en) | A kind of hair clip Mdification primer of combination universal fluorescent primer | |
| CN111575386B (en) | Fluorescent composite amplification kit for detecting human Y-SNP locus and application thereof | |
| CN110747280B (en) | TE mark related to cold adaptability of horse and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190607 |