CN108715888A - A kind of homogeneous detecting systems of PCR based on FRET and its application - Google Patents
A kind of homogeneous detecting systems of PCR based on FRET and its application Download PDFInfo
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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
- C12Q1/6858—Allele-specific amplification
Abstract
The present invention provides the tailed primer of the connection sequence label of a kind of homogeneous detecting system of the PCR based on FRET and its application, the homogeneous detection systems of the PCR, including at least one fluorescence or quenched label, you can to start the primer of DNA synthesis；With sequence label it is complementary be quenched or the oligonucleotide sequence of fluorescent marker and reversed amplimer.The present invention by quantitative fluorescent PCR instrument can timely observation signal, and be instantly obtained result at the end of reaction.
Description
Technical field
The present invention relates to genetic test fields, more particularly to a kind of homogeneous detecting systems of PCR based on FRET and its answer
With.
Background technology
SNP detections are a kind of most genetic tests of current detection demand.SNP classifying methods are in different poses and with different expressions, but with
The emergence of new technology, new method and instrument, some methods have not used, or are used merely to do complementary test
Card on some methods are very tall and big, but is not suitable for a wide range of use.
Single nucleotide polymorphism refers to the polymorphism of DNA sequence dna caused by the change of single base on genome.Mainly
Refer to conversion and transversion, also includes few insertion or missing.SNP is most common in hereditary variation, accounts for known polymorphism
90% or more, it is widely present in human genome.Since its distribution is wide, stablize the feature of heredity, many disease susceptibilities or
Drug response difference is all closely related with it, so, it is applied to molecule diagnosis, new drug development, individuation more and more at present
Medication, clinical examination etc..
Taqman sonde methods, only carry out parting by a kind of mechanism of annealing temperature competition, and Arms sonde methods are unable to real-time fluorescence inspection
It surveys.
7615620 B2 of United States Patent (USP) US disclose a kind of PCR detection system, and wherein Kasp methods detection SNP site needs 7
Oligonucleotides, and mainly terminal SNP partings, are not suitable for real-time fluorescence SNP partings.
Invention content
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of PCR based on FRET is homogeneous
Detecting system and its application need 7 oligonucleotides for solving Kasp methods detection SNP site in the prior art, and main
The problems such as being terminal SNP partings, not being suitable for real-time fluorescence SNP partings.
In order to achieve the above objects and other related objects, it is homogeneous to provide a kind of PCR based on FRET for first aspect present invention
Detecting system includes the tailed primer for being connected with sequence label of at least one fluorescence or quenched label, you can to start DNA conjunctions
At primer；With the sequence label it is complementary be quenched or the oligonucleotide sequence of fluorescent marker and reversed amplimer.
In some embodiments of the invention, the length of the sequence label is 9-125bp.
In some embodiments of the invention, the length of oligonucleotide sequence is 9-125bp.
In some embodiments of the invention, the complimentary fashion of the sequence label and the oligonucleotide sequence includes just
To complementary, reverse complemental.
In some embodiments of the invention, the mark position of fluorescence or quenching group is the arbitrary alkali of the sequence label
Base location, the labelling groups on labelling groups and oligonucleotide sequence on tailed primer are close to each other.
In some embodiments of the invention, fluorescence marker groups be selected from FAM, TET, HEX, VIC, JOE, TAMRA,
At least one of ROX, TEXAS-RED, CY3, CY5 etc..The fluorophor for having similar launch wavelength with aforementioned group is suitable
For the present invention.
In some embodiments of the invention, quenched label group be selected from BHQ0, BHQ1, BHQ2, BHQ3, TAMRA, MGB,
DABCYL, ECLIPSE etc. are therewith similar at least one of quenching group of absorbing wavelength.There is similar transmitting with aforementioned group
The fluorophor of wavelength is suitable for the present invention.
In some embodiments of the invention, the tailed primer is two, containing as follows in one of tailed primer
Sequence label：TGGACTACCATAGATGTGCTGC (SEQ ID NO.6) contains following label sequence in another tailed primer
Row：ACTTCACCGGTCTGGCGGGTTCA(SEQ ID NO.7).Sequence label is tailing sequence.Before sequence label is not limited to
Two sequences are stated, according to the difference of cls gene to be checked, may be designed in other sequence labels.
In some embodiments of the invention, the tailed primer for being connected with sequence label of the fluorescent marker includes G-
The nucleotide sequence of F1-25, A-F1-25, the G-F1-25 are as follows：
5’-FAM-TGGACTACCATAGATGTGCTGCACTATTTTCTTGACCCCTACTTACG-3’(SEQ ID
NO.1)；Single underscore partial sequence is sequence label.
The nucleotide sequence of the A-F1-25 is as follows：
5’-HEX-ACTTCACCGGTCTGGCGGGTTCAACTATTTTCTTGACCCCTACTTACA-3’(SEQ ID
NO.2)；Single underscore partial sequence is sequence label.
The tailed primer sequence of the connection sequence label of the quenched label includes that F is quenched, H is quenched, the core that F is quenched
Nucleotide sequence is as follows：
5'-GCAGCACATCTATGGTAGTCCA-BHQ1-3'(SEQ ID NO.3)；
The nucleotide sequence that H is quenched is as follows：
5'-TGAACCCGCCAGACCGGTGAAGT-BHQ1-3'(SEQ ID NO.4)；
The nucleotide sequence of the reversed amplimer is as follows：
TAGTAGACAACATACGACCAC(SEQ ID NO.5)。
Second aspect of the present invention provides application of the homogeneous detecting systems of above-mentioned PCR in single nucleotide polymorphism detection.
Monitoring, the present invention can be used for specific target sequence in real-time fluorescence PCR to PCR processes in real time after multiple cycle
Augmentation detection.Applicable real-time fluorescence PCR instrument includes but not limited to：Applied Biosystems (ABI) company 7300,
7500, LightCycler2.0, LightCycler480 of 7700, Roche companies, the TL998A etc. of Xi'an Tian Long companies.
PCR detection process after PCR anneals only by using being monitored.
The present invention is not limited to ApoE gene, SNP partings, gene delection and pathogen detections.PCR was detected
Journey after PCR anneals only by using being monitored.
As described above, a kind of homogeneous detecting systems of PCR based on FRET and its application of the present invention, have below beneficial to effect
Fruit：The present invention is separately connected one and the unmatched fluorescence tail of template in the ends 5' of two primers of ARMS-PCR, and respectively
Synthesize a corresponding reverse complemental primer sequence.Designed fluorescence labels sequence is with tested cls gene difference source or not
It is completely homologous.The ends tail 5' carry out fluorescent base single group mark (FAM or HEX, be not limited to both), therewith the complementary ends sequence 3'
(BHQ1 or BHQ2 etc., be not limited to both) is marked with quenching group, designed two sequence label melting temperature (Tm
Value), G/C content it is close, it is ensured that unwinding state is close at the same temperature.The ends the 3' base of the fluorescent primer of this two tailings point
It is not combined with the allele of wild type, saltant type.If amplimer 3 ' holds base to exactly match, fluorescent primer will be suitable
Profit extends into template, and tail, which is quenched, can dissociate and discharge corresponding fluorescence；If amplimer 3 ' holds base to form mispairing, extend just
Can be blocked, fluorescent primer still with primer is quenched is combined, without the release of corresponding fluorescence.It can be with by quantitative fluorescent PCR instrument
Timely observation signal, and it is instantly obtained result at the end of reaction.
Description of the drawings
Fig. 1 a-1e are shown as the single primer amplification principle schematic of the present invention.
Fig. 2 a-2e are shown as the SNP Genotyping testing principle schematic diagrames of the present invention.
Fig. 3 is shown as carrying the plasmid map of rs662-A target fragments in the embodiment of the present invention.
Fig. 4 is shown as carrying the plasmid map of rs662-G target fragments in the embodiment of the present invention.
Fig. 5 is shown as single group fluorescent primer amplification curve diagram in the embodiment of the present invention 1.
Fig. 6 is shown as double fluorescent primer group amplification curve diagrams of plasmid GG in the embodiment of the present invention 2.
Fig. 7 is shown as double fluorescent primer group amplification curve diagrams of plasmid AA in the embodiment of the present invention 2.
Fig. 8 is shown as double fluorescent primer group amplification curve diagrams of plasmid GA in the embodiment of the present invention 2.
Specific implementation mode
Illustrate that embodiments of the present invention, those skilled in the art can be by this specification below by way of specific specific example
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.
It should be clear that in the following example not specifically dated process equipment or device be all made of conventional equipment in the art or
Device；All pressure values and range all refer to absolute pressure.
In addition, it should also be understood that, one or more method and step mentioned in the present invention does not repel before and after the combination step
It can also be inserted into other methods step there may also be other methods step or between these explicitly mentioned steps, unless separately
It is described；It should also be understood that the combination connection relation between one or more equipment/device mentioned in the present invention is not repelled
The front and back two equipment/devices specifically mentioned there may also be other equipment/device or at these of the unit equipment/device it
Between can also be inserted into other equipment/device, unless otherwise indicated.Moreover, unless otherwise indicated, the number of various method steps is only
Differentiate the convenient tool of various method steps, rather than to limit the ordering of various method steps or limiting the enforceable model of the present invention
It encloses, relativeness is altered or modified, and without material changes in technical content, when being also considered as, the present invention is enforceable
Scope.
The method that SNP site is quickly detected about one-step method, such as double TaqMan probe methods, can detect in time, but only
The specificity of detection can be improved by the binding specificity with template.And for by with template joint efficiency and extension mechanism knot
The ARMS-PCR methods of conjunction need second detecting step of increase that can just obtain testing result, extend needed for detection again in this way
The time wanted.The advantages of existing TaqMan of the present invention quickly goes out result, and have the advantages that ARMS method specificity.This stopped pipe side
Method detects sample, it is only necessary to which or so two hours can go out as a result, not only easy to operate, also shorten detection time.Finally
The result arrived is also more intuitive, can see that figure compares, be easy to obtain a result.
Single primer amplification application
As shown in Fig. 1 a- Fig. 1 e, a sequence label, abbreviation tailed primer are connected in 5 ' ends of PCR primer.This mark
It signs sequence to mismatch with template, one fluorophor of label (common FAM) on sequence label.And that there are one is reversed with this label
Complementary label, complementary label connection quenching group (common BHQ1 labels).
When two tag complements, fluorescence labels and tag complement specific bond is quenched, fluorescence is quenched.Tailed primer exists
After the lower startup PCR of archaeal dna polymerase effect, tailed primer participates in the synthesis of the new chains of DNA, mixes in new template, reverse primer is with new
Chain is the new template double-strand of templated synthesis.Tailed primer is detached with label is quenched, fluorescence release, the fluorescence signal and PCR of generation
The quantity of product is directly proportional.
The application of SNP Genotypings
As shown in Fig. 2 a- Fig. 2 e, increases by one group of tailed primer on the basis of first application and label is quenched.Two kinds add
The sequence label of tail primer marks different fluorescence (common FAM and HEX), and two groups are quenched label and can mark and identical base are quenched
Group (common BHQ1) or different quenching group (BHQ1 or BHQ2).
3 ' the two different genotype bases in end oppositional allele site of two tailed primers.When tailed primer 3 '
When end is exactly matched with template, PCR could start；When mismatch, PCR is blocked.Two groups of tailed primers start respectively corresponding
Template, in the double-strand for mixing new template, and release corresponding fluorescence, two kinds of fluorescence signals of generation with it is corresponding
The quantity of PCR product is directly proportional, accordingly the genotype of judge templet.
Embodiment 1
The amplification experiment of single group fluorescent primer, the present embodiment are inserted into human gene to structure plasmid and are detected
1 primer sequence table of table
The both ends mutational site R (R=A/G) total 300bp of number of base is intercepted, the two bases is directed to respectively and synthesizes two matter
Grain：Rs662-A and rs662-G, as the standard Quality Control of this experiment contrast, plasmid map is as shown in Figure 3 and Figure 4.Carrier is by upper
Hai Xuguan biotechnologies Development Co., Ltd provides, cloning vector：PES, resistance：Ampicillin, insertion point：EcoRV, bacterium
Strain：DH5 α, sequence is as shown in SEQ ID NO.5.
Rs662-A DNA sequence dnas are as follows：
TAATAATCCTGTAATGTTCAATACCTTCACCTTATATATTATGTGTGTATGTTTTAATTGCAGTTTGAATGATATTG
TTGCTGTGGGACCTGAGCACTTTTATGGCACAAATGATCACTATTTTCTTGACCCCTACTTACAATCCTGGGAGATG
TATTTGGGTTTAGCGTGGTCGTATGTTGTCTACTATAGTCCAAGTGAAGTTCGAGTGGTGGCAGAAGGATTTGATTT
TGCTAATGGAATCAACATTTCACCCGATGGCAAGTATGTGAACTCTCTGAAATGTAGTGGATTTACTCA(SEQ ID
NO.8)。
Rs662-G DNA sequence dnas are as follows：
TAATAATCCTGTAATGTTCAATACCTTCACCTTATATATTATGTGTGTATGTTTTAATTGCAGTTTGAATGATATTG
TTGCTGTGGGACCTGAGCACTTTTATGGCACAAATGATCACTATTTTCTTGACCCCTACTTACGATCCTGGGAGATG
TATTTGGGTTTAGCGTGGTCGTATGTTGTCTACTATAGTCCAAGTGAAGTTCGAGTGGTGGCAGAAGGATTTGATTT
TGCTAATGGAATCAACATTTCACCCGATGGCAAGTATGTGAACTCTCTGAAATGTAGTGGATTTACTCA(SEQ ID
NO.9)。
With the homozygous plasmid template of substance fluorescent primer group (selection HEX groups) amplification various concentration, (plasmid carries mesh
Segment rs662-A, be expressed as PES+rs662-A), plasmid concentration is 10 respectively6copies/μL、105copies/μL、
104Copies/ μ L, Tm are 58 DEG C selected, reaction system such as the following table 2：
The amplification experimental system of 2 HEX fluorescent primer groups of table
Response procedures are as follows：95 DEG C of pre-degeneration 60s, 95 DEG C of denaturation 15s, 58 DEG C of annealing 30s, 50 recycle.
When Fig. 5 is shown as Tm=58 DEG C, HEX fluorescent primer groups, plasmid concentration and amplification curve change curve (wherein matter
Grain concentration A=1 × 106Copies/ μ L, B=1 × 105Copies/ μ L, C=1 × 104copies/μL.On the grand instrument in day,
FAM signals indicate that HEX signals are indicated with green curve with blue curve).
With the reduction of template concentrations, curve ct values gradually increase.Fluorescence signal intensity is between 4500-5500.Pass through
Δ Ct is calculated, as a result such as table 3, the single group fluorescent primer of the method can also do relative quantification by comparing standard items.
3 HEX fluorescent primer groups of table expand the CT values of various concentration plasmid
Embodiment 2
SNP detections are done to the sites rs662
Primer sequence is as shown in table 4 below.
4 primer sequence table of table
The present embodiment is detected human leucocyte gene, and reaction system is as shown in table 5 below：
Table 5 detects reaction system composition when human leucocyte gene
PCR response procedures are as follows：95 DEG C of pre-degeneration 60s, 95 DEG C of denaturation 15s, 56 DEG C of annealing 30s, 50 recycle.
Fig. 6 is shown as amplification curve diagram of the double fluorescent primer groups of plasmid GG on quantitative fluorescent PCR instrument.FAM institutes are right
The blue curve answered is very high, and the green curve corresponding to HEX is very low, it can be seen that and only FAM has apparent amplification, belongs to GG types,
It can be with parting.
Fig. 7 is shown as amplification curve diagram of the double fluorescent primer groups of plasmid AA on quantitative fluorescent PCR instrument.FAM institutes are right
The blue curve answered is very low, and the green curve corresponding to HEX is very high, it can be seen that and only HEX has apparent amplification, belongs to AA types,
It can be with parting.
Fig. 8 is shown as amplification curve diagram of the double fluorescent primer groups of plasmid GA on quantitative fluorescent PCR instrument.FAM institutes are right
The blue curve answered is overlapped with the green curve corresponding to HEX, it can be seen that the two has apparent fluorescence signal to discharge, and belongs to
GA types, can be with parting.
In conclusion the present invention the ends 5' of two primers of ARMS-PCR be separately connected one it is unmatched glimmering with template
Light tail, and it is respectively synthesized a corresponding reverse complemental primer sequence.Designed fluorescence labels sequence be detected
Gene is not homologous or not exclusively homologous.The ends tail 5' carry out fluorescent base single group label (FAM or HEX, be not limited to both), with
Complementation the ends sequence 3' with quenching group mark (BHQ1 or BHQ2 etc., be not limited to both), designed two mark
It is close to sign sequence melting temperature (Tm values), G/C content, it is ensured that unwinding state is close at the same temperature.The fluorescence of this two tailings
The ends the 3' base of primer is combined with the allele of wild type and saltant type respectively.If amplimer 3 ' holds complete of base
Match, fluorescent primer will smoothly extend into template, and tail, which is quenched, can dissociate and discharge corresponding fluorescence；If amplimer 3 ' holds alkali
Base forms mispairing, and extension will be blocked, fluorescent primer still with primer is quenched is combined, without the release of corresponding fluorescence.By glimmering
Fluorescent Quantitative PCR instrument can timely observation signal, and be instantly obtained result at the end of reaction.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology can all carry out modifications and changes to above-described embodiment without violating the spirit and scope of the present invention.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should by the present invention claim be covered.
SEQUENCE LISTING
<110>Wang Weidong
<120>A kind of homogeneous detecting systems of PCR based on FRET and its application
<130> 183581
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 47
<212> DNA
<213> Artificial
<220>
<223> G-F1-25
<400> 1
tggactacca tagatgtgct gcactatttt cttgacccct acttacg 47
<210> 2
<211> 48
<212> DNA
<213> Artificial
<220>
<223> A-F1-25
<400> 2
acttcaccgg tctggcgggt tcaactattt tcttgacccc tacttaca 48
<210> 3
<211> 22
<212> DNA
<213> Artificial
<220>
<223>F is quenched
<400> 3
gcagcacatc tatggtagtc ca 22
<210> 4
<211> 23
<212> DNA
<213> Artificial
<220>
<223>H is quenched
<400> 4
tgaacccgcc agaccggtga agt 23
<210> 5
<211> 21
<212> DNA
<213> Artificial
<220>
<223> AG-R1-21
<400> 5
tagtagacaa catacgacca c 21
<210> 6
<211> 22
<212> DNA
<213> Artificial
<220>
<223>Sequence label 1
<400> 6
tggactacca tagatgtgct gc 22
<210> 7
<211> 23
<212> DNA
<213> Artificial
<220>
<223>Sequence label 2
<400> 7
acttcaccgg tctggcgggt tca 23
<210> 8
<211> 300
<212> DNA
<213> Artificial
<220>
<223> rs662-A
<400> 8
taataatcct gtaatgttca ataccttcac cttatatatt atgtgtgtat gttttaattg 60
cagtttgaat gatattgttg ctgtgggacc tgagcacttt tatggcacaa atgatcacta 120
ttttcttgac ccctacttac aatcctggga gatgtatttg ggtttagcgt ggtcgtatgt 180
tgtctactat agtccaagtg aagttcgagt ggtggcagaa ggatttgatt ttgctaatgg 240
aatcaacatt tcacccgatg gcaagtatgt gaactctctg aaatgtagtg gatttactca 300
<210> 9
<211> 300
<212> DNA
<213> Artificial
<220>
<223> rs662-G
<400> 9
taataatcct gtaatgttca ataccttcac cttatatatt atgtgtgtat gttttaattg 60
cagtttgaat gatattgttg ctgtgggacc tgagcacttt tatggcacaa atgatcacta 120
ttttcttgac ccctacttac gatcctggga gatgtatttg ggtttagcgt ggtcgtatgt 180
tgtctactat agtccaagtg aagttcgagt ggtggcagaa ggatttgatt ttgctaatgg 240
aatcaacatt tcacccgatg gcaagtatgt gaactctctg aaatgtagtg gatttactca 300
<210> 10
<211> 2692
<212> DNA
<213> Artificial
<220>
<223> PES
<400> 10
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatccg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggc acccggggat 420
cttccagaga tatcgtcgaa cggcaggcgt gcaaacttgg cgtaatcatg gtcatagctg 480
tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 540
aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 600
ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 660
gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 720
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 780
tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 840
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 900
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 960
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 1020
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 1080
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 1140
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 1200
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 1260
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 1320
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 1380
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 1440
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 1500
tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 1560
tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 1620
tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 1680
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 1740
ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1800
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1860
gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1920
agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1980
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 2040
tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 2100
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 2160
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 2220
cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 2280
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 2340
ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 2400
actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 2460
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 2520
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 2580
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 2640
attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc 2692
Claims (8)
1. a kind of homogeneous detecting systems of PCR based on FRET, including at least one fluorescence or quenched label are connected with label sequence
The tailed primer of row, with sequence label it is complementary be quenched or the oligonucleotide sequence of fluorescent marker and reversed amplimer.
2. the homogeneous detecting systems of PCR according to claim 1, it is characterised in that：The length of the sequence label is 9-
125bp。
3. the homogeneous detecting systems of PCR according to claim 1, it is characterised in that：The sequence label and the few nucleosides
The complimentary fashion of acid sequence includes positive complementary, reverse complemental.
4. the homogeneous detecting systems of PCR according to claim 1, it is characterised in that：Fluorescence marker groups be selected from FAM, TET,
At least one of HEX, VIC, JOE, TAMRA, ROX, TEXAS-RED, CY3, CY5；Quenched label group be selected from BHQ0,
At least one of BHQ1, BHQ2, BHQ3, TAMRA, MGB, DABCYL, ECLIPSE.
5. the homogeneous detecting systems of PCR according to claim 1, it is characterised in that：The mark position of fluorescence or quenching group
For the arbitrary base positions of the sequence label, the labelling groups on tailed primer and the label base on the oligonucleotide sequence
Group is close to each other.
6. the homogeneous detecting systems of PCR according to claim 1, which is characterized in that the tailed primer is two, wherein one
Contain following sequence label in a tailed primer：TGGACTACCATAGATGTGCTGC (SEQ ID NO.6), another tailing draws
Contain following sequence label in object：ACTTCACCGGTCTGGCGGGTTCA(SEQ ID NO.7)。
7. the homogeneous detecting systems of PCR according to claim 1, which is characterized in that the connection label sequence of the fluorescent marker
The tailed primer sequence of row includes G-F1-25, A-F1-25, and the nucleotide sequence of the G-F1-25 is as follows：
5’-FAM-TGGACTACCATAGATGTGCTGCACTATTTTCTTGACCCCTACTTACG-3'(SEQ ID NO.1)；
The nucleotide sequence of the A-F1-25 is as follows：
5’-HEX-ACTTCACCGGTCTGGCGGGTTCAACTATTTTCTTGACCCCTACTTACA-3’
(SEQ ID NO.2)；
The tailed primer sequence of the connection sequence label of the quenched label includes that F is quenched, H is quenched, the nucleotide that F is quenched
Sequence is as follows：
5'-GCAGCACATCTATGGTAGTCCA-BHQ1-3'(SEQ ID NO.3)；
The nucleotide sequence that H is quenched is as follows：
5'-TGAACCCGCCAGACCGGTGAAGT-BHQ1-3'(SEQ ID NO.4)；
The nucleotide sequence of the reversed amplimer is as follows：
TAGTAGACAACATACGACCAC(SEQ ID NO.5)。
8. according to application of the homogeneous detecting systems of PCR described in claim 1-7 any one in single nucleotide polymorphism detection.
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| CN114540345A (en) * | 2021-11-03 | 2022-05-27 | 武汉蓝沙医学检验实验室有限公司 | Labeled fluorescent probe with hairpin structure and fluorescence detection method |
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