CN107400722A - A kind of competitive real-time fluorescence PCR SNP probes for detecting human genome - Google Patents
A kind of competitive real-time fluorescence PCR SNP probes for detecting human genome Download PDFInfo
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- CN107400722A CN107400722A CN201710829135.2A CN201710829135A CN107400722A CN 107400722 A CN107400722 A CN 107400722A CN 201710829135 A CN201710829135 A CN 201710829135A CN 107400722 A CN107400722 A CN 107400722A
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Classifications
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Abstract
A kind of competitive real-time fluorescence PCR SNP probes for detecting human genome, are related to SNP probe.With secondary structure, comprising with complementary series and not possessing the fluorescence probe of water-disintegrable semicircular structure and fully-complementary sequence completely, it is that SNP is located at 5 ' ends of fluorescence probe, and in 3 ' 3~5 bases of end design of fluorescence probe, combine 5 ' ends of itself and fluorescence probe, so as to form secondary structure.In addition, hold neighbouring additional 3~7 not bases with probe self-complementary 3 '.This special construction is greatly improved water-disintegrable, reduction autofluorescent background signal, and so as to improve real-time PCR sensitivity, increasing the specificity of probe reduces false positive rate.Sensitivity and the specificity of human genome SNP detections are greatly improved, reduces false positive rate, while economical and convenient again.
Description
Technical field
The present invention relates to SNP (Single Nucleotide Polymorphism, SNP) probe, especially
It is to be related to a kind of competitive real-time fluorescence PCR SNP probes for detecting human genome.
Background technology
SNP (Single Nucleotide Polymorphism, SNP), is primarily referred to as in genome water
As the DNA sequence polymorphism caused by the variation of single nucleotide acid on flat.It is most common one in the heritable variation of the mankind
Kind, account for more than the 90% of all known polymorphisms.SNP is widely present in human genome, average every 500~1000 bases
Centering just has 1, estimates that its sum is even more more up to 3,000,000.
The polymorphism that SNP is showed relates only to the variation of single base, and this variation can be by the conversion of single base
(Transition) or transversion (Transversion) is caused, also can be caused by the insertion of base or missing.It is but usually said
SNP do not include latter two situation.
Theoretically, SNP had both been probably two equipotential polymorphisms, it is also possible to 3 or 4 equipotential polymorphisms, but in fact,
Both are very rare afterwards, can almost ignore.Therefore, usually said SNP is two equipotential polymorphisms.This variation may
It is conversion (C T, being then G A on its complementary strand), it is also possible to transversion (C A, G T, C G, A T).The incidence of conversion is total
It is apparently higher than other several variations, the SNP with conversion form variation accounts for 2/3, and the occurrence probability of other several variations is similar.
Wang etc. research also demonstrates this point.Why high the probability of conversion is, it may be possible to because the cytimidine on CpG dinucleotides
Residue is the site most easily undergone mutation in human genome, wherein most of methylate, can spontaneously slough amino and
Form thymidine.
In genomic DNA, any base is likely to occur variation, therefore SNP is both possible in gene order,
It is possible on the non-coding sequence beyond gene.Generally speaking, the SNP in code area (coding SNP, cSNP) compares
It is less because in extron, its aberration rate only and surrounding sequence 1/5.But it has weight in genetic disease research
Meaning is wanted, therefore cSNP research is more concerned.
At present, detecting SNP common method includes PCR- direct sequencings, PCR- pyrosequencings method, quantitative fluorescent PCR
Method, PCR- gene chips, PCR- electrophoretic analysis, PCR- high-resolution melting curves method, ApoE gene method,
A variety of methods such as PCR- RFLPs method, in situ hybridization (ISH), the principle and advantage and disadvantage of every kind of method are such as
Under：
1) PCR- direct sequencings
Also referred to as PCR-Sanger is sequenced, and this method is based on bi-deoxyribose nucleic acid (ddNTP) chain termination method, according to nucleosides
Acid start to extend in a certain fixing point, at random at a certain particular bases terminate, due to each base of incorporation carried out it is glimmering
Signal, therefore generate with the serial nucleic acid fragment of the different length of A, T, C, G four groups of differences, one base terminated；Pass through
The base sequence of determined nucleic acid is read after these fragments of capillary electrophoresis separation.The sequencing of Sanger methods is the warp of DNA sequence analysis
Allusion quotation method.Because this method can directly read DNA sequence, therefore it is considered as the goldstandard of Genotyping.
The operating process of PCR-Sanger PCR sequencing PCRs mainly includes PCR amplifications and PCR primer purifying, sequencing reaction, sequencing
With four key steps of interpretation of result.Need to set negative control and positive quality control product during analysis.This method belongs to qualitative detection,
Advantage is that sequencing length is longer, it is possible to find new variant sites.Main deficiency：Sensitivity is not high, is especially carrying out tumor group
When knitting somatic mutation detection, when target gene mutant proportion is less than 20% in tissue, in fact it could happen that false negative result；To examination
Agent and instrument have particular/special requirement, are not easy to popularize；Complex operation, cost is of a relatively high, and speed is slow, flux is low.
2) PCR- pyrosequencings method
This method is the enzyme cascade chemiluminescence reaction in the same reaction system by 4 kinds of enzymatics.Experiment need to design one
The sequencing primer of bar biotin labeling, after primer and single-stranded template DNA annealing, in archaeal dna polymerase, ATP sulfurylases, fluorescence
Under the synergy of plain 4 kinds of enzymes of enzyme and apyrase, by the polymerization of each dNTP on primer and first order fluorescence
The release coupling of signal is got up, and by detecting release and the intensity of fluorescence, reaches the purpose of the real time measure DNA sequence dna.Required examination
Agent includes sample processing reagent, nucleic acid amplification agents, single-stranded template reagent preparation, pyrosequencing reagent and positive quality control product five
Major class.Required instrument is PCR instrument and pyrosequencing instrument.To avoid false positive and false negative result, positive matter should be strictly distinguished
The use of control product and reaction reagent, prevent because reagent contamination causes false positive.
The major advantage of this method：Detection sensitivity is higher, to somatic mutation and the achievable quantitative detection such as methylate；
Accurately and reliably, flux is higher for parting, and experimental design is flexible, it is possible to find new mutation or hereditary variation.Major defect：To reagent and
Instrument has particular/special requirement, is not easy to popularize；Detection sensitivity is limited, in tumor tissues low abundance somatic mutation (<3%) hold
Easily there is false negative；Sequencing length individual base only more than 10, it is impossible to analyze long segment.
3) real-time fluorescence PCR method
According to the difference of Cleaning Principle, real-time fluorescence PCR method can be divided into two kinds of sonde method and non-sonde method, the former using with
The probe (Taqman and molecular beacon) of target sequence specific hybridization indicates the increase of amplified production, the latter using fluorescent dye or
The primer of particular design indicates the increase of amplified production.Taqman sonde methods combine 5 ' end nucleases and fluorescence simultaneously
Etc. technology, it uses 4 oligonucleotide chains in course of reaction, and two of which is allele-specific probe, and two are drawn for PCR
Thing.Two probes can be complementary with saltant type and wild-type template respectively, and application contains reporter group and quenching group respectively at its both ends
Dyestuff be marked, the reporter group fluorescent dye of two probes is different.When carrying out SNP detections, the annealing of PCR amplifications
Process causes probe and template to hybridize to combine, and when at primer extend to probe, 5 ' end 5 prime excision enzyme activities of archaeal dna polymerase will be visited
5 ' end reporter groups of pin are cut off from probe, are allowed to separate with quenching group, so as to discharge corresponding fluorescence, without
The probe of pairing is remained in that completely without fluorescing.Different allele probes because the fluorescent dye of mark is different,
Therefore fluoresce signal difference, can pass through the genotype of the detection judgement sample to fluorescence signal.
Real-time fluorescence PCR method high sensitivity, parting is accurate, simple and efficient to handle, and instrument is easily popularized, easy to spread
Use.But this method flux is not high, probe cost is higher, and the testing cost of Single locus is relevant with sample size, and sample size is smaller,
Cost is higher.This method is primarily adapted for carrying out parting to a small amount of site, large sample.CFDA approveds CYP2C9, VKORC1 at present
Etc. the PCR- fluorescence detection reagent kits of several genes polymorphic detection.
4) PCR- gene chips
It is regularly arranged and is fixed on holder using specific oligonucleotide fragment as probe by this method, so
Sample DNA is expanded by PCR afterwards, the program such as fluorescence labeling, by base pairing principle and chip hybridization, then pass through fluoroscopic examination
System is detected and analyzed to the fluorescence signal on chip, so as to obtain the genotype information of individual rapidly.Genetic chip point
The operating process of type method includes PCR nucleic acid amplifications, hybridization, chip scanning and interpretation of result.When this method is used for DNA Genotypings
Belong to qualitative detection, sensitivity is 50ng/ μ L.Gene chips need to set negative control and positive quality control product when analyzing.Using
It is ensured that vibration mixing before reagent preserves on request before Kaifeng, each component liquid uses, miscellaneous during genechip detection kit
Operation of handing over and develop a film must be carried out under the conditions of lucifuge, and with reference to that need to be kept in dark place, chip pays attention to when being loaded for PCR reaction solutions and positioning
Liquid is paved with whole reaction zone, but can not overflow, bubble can not occur, with anti-cross-contamination.Its major advantage is can be simultaneously
The base sequence of multiple SNP sites to be measured is detected.China's CFDA approveds are a variety of to be used for drug metabolic enzyme and medicine work
Tried with target gene, such as genetic chip of ALDH2, CYP2C9, CY2C19, CYP2D6, ADR1, ACE, VKORC1 polymorphic detection
Agent box.
5) PCR- electrophoretic analysis
This method refers to enter target gene fragment to be analyzed performing PCR amplification, and passes through agarose gel electrophoresis or hair
Cons electrophoresis is analyzed, and Genotyping is carried out to gene polymorphism sites according to the size of PCR primer.This method belongs to qualitative inspection
Survey, and be only used for detecting known pleomorphism site, it is impossible to identify unknown polymorphism.Agarose electrophoresis is applied to
The insertion deletion longer to fragment detects, such as ACE insertion deletions；Capillary electrophoresis is suitable to shorter
Insertion deletion such as UGT1A1*28 polymorphisms and microsatellite instability (MSI) detected.Need to build during PCR
Vertical positive quality control product and negative quality-control product, need carry out the judgement of clip size with molecular weight marker simultaneously during electrophoretic analysis.When
Molecular weight marker reaction tube without band or when there is weaker band, it is possible the reason for include loading wells leak, fluorescent dye not
Enough or failure, electrophoresis time is long or voltage is excessive.The advantages of this method is that cost is low, can be carried out in common lab；Lack
Point is to be only suitable for carrying out qualitative determination to DNA insertion/deletions or fusion, it is impossible to be used in SNP detection.
6) PCR- high-resolution melting curve (HRM) method
This method carries out Genotyping by the melting curve analysis reacted PCR.The melting curve of PCR amplifications depends on
Its extension increasing sequence, the difference of a base all can cause the melting temperature of double-stranded DNA to change in sequence.The application of HRM methods is real
When quantitative real time PCR Instrument monitor this trickle temperature change, it is determined that with the presence or absence of mutation in the purpose fragment expanded, so as to
For Genotyping.Using saturated fluorescence dyestuffs such as LC Green, such dyestuff is reacted PCR in saturated concentration for HRM analyses
Unrestraint acts on, therefore can be used with high concentration, so as to all with reference to the ditch in DNA double helical structure.In the change of double-stranded DNA
Property process the rearrangement of fluorescence molecule is not present, its specificity is increased dramatically, and therefore, the trickle change of melting curve can be anti-
Reflect the difference of base in amplified fragments.Genotyping, which is carried out, using this method belongs to qualitative analysis.
This method is easy to operate, quick, flux is big, use cost is low, result is accurate, is advantageously implemented stopped pipe operation,
Can be determined during DNA methylation assay according to melting curve the height of methylation.The shortcomings that this method is：It can not exclude to treat
Survey emerging hereditary variation in nucleic acid；Because single base mutation causes the change of DNA melting temperatures very small, this method pair
The sensitivity of instrument and resolution ratio have higher requirements.
7) ApoE gene (Allele-specific PCR, AS-PCR)
Also known as amplification refractory mutation system PCR (Amplification Refractory Mutation System
PCR, ARMS-PCR).The general principle of the technology：Because Taq archaeal dna polymerases lack the 5 prime excision enzyme activity at 3 ' to 5 ' ends, 3 ' ends
The base of mispairing can cause primer extend to slow, and when mispairing reaches to a certain degree, primer extend will terminate, cannot spy
The pcr amplification product of different length, it is on the contrary then have so as to prompt base of the template DNA with the pairing of the end of primer 3 '.Therefore, AS-
PCR reactions need the primer and a shared reverse primer of two allele specifics, and two non-specific primers are at 3 ' ends
With template mispairing, but other parts base sequence is just the same.When 3 ' ends of only primer are matched completely with template, PCR amplifications
It can just carry out.PCR primer can carry out analysis and the judgement of genotype by gel electrophoresis.This method can also determine with real-time fluorescence
Amount PCR combines carry out Genotyping.This method can be used for detecting various types of SNP, and its advantage is high sensitivity, special
It is not suitable for detecting the somatic mutation in tumor tissues；Shortcoming is that false positive rate is higher.
8) PCR- RFLPs method
RFLP method (RFLP) is a kind of method based on digestion principle, is to be used for gene earliest
One of classical way of parting, still it is widely adopted now.This method is based primarily upon some restriction enzymes can specificity
Identify a certain particular sequence and structural DNA, and the principle sheared to it.The generally recognized double-stranded DNA of restriction enzyme
A certain particular sequence, and in ad-hoc location or nearby cut off double-stranded DNA, so as to produce shorter DNA fragmentation.Due to limitation
The stringency of property endonuclease recognition sequence, the change of a base can result in the disappearance of digestion activity.Using this characteristic,
If treat the base sequence of the SNP site of parting on the recognition site of a certain restriction enzyme, it will to cause the enzyme only to it
In a kind of allele there is digestion activity.Therefore, can when being pointed to the SNP progress partings of restricted digestion recognition site
To be incubated using the PCR primer comprising the site with corresponding restriction enzyme.The later product of digestion carries out electrophoresis,
And Genotyping is carried out according to the size of digestion products fragment.This method does not need any probe, it is not required that special instrument
Device equipment, cost is relatively low, and experimentation is simple, workable.But shortcoming is it is also obvious that mainly flux is too low, great Liang Fen
Workload is big during type, and is only applicable to part SNP partings.
9) in situ hybridization (ISH) method
ISH methods are with various human body specimens, including cytology and histological specimen (formalin prepared by corresponding experimental method
Fixed FFPE) target is used as, molecule hybridization is carried out using target DNA probe and the target, so as to detect the target base of correlation
Because of exception.ISH technologies can be divided into bright visual field in situ hybridization and FISH (FISH) according to the type of probe label.
The target of ISH methods detection has complete nucleus, without carrying out the extraction of nucleic acid.Its specific methodology principle is shown in《It is in situ
Hybridize (ISH) guide》.In drug metabolic enzyme and target gene detection, ISH methods are mainly used in determining gene magnification and gene lacks
Lose abnormal.
Bibliography：
[1]Deng YM,Spirason N,Iannello P,et al.A simplified Sanger sequencing
method for full genome sequencing of multiple subtypes of human influenza A
viruses.J Clin Virol.2015Jul；68:43-8.
[2]Li R,Li Y,Fang X,et al.SNP detection for massively parallel whole-
genome resequencing.[J].Genome Research,2009,19(6):1124。
[3] Wang Mojin, Zhou Zongguang, Wang Ling, is waited to use TaqMan probe real-time fluorescence PCR technology for detection AKAP10 genes
2073A/G SNPs (J) Sichuan University's journals (medicine), 2009,40 (2):275-278.
[4]Multicolor molecular beacons for allele discriminationSanjay
Tyagi1,*,Diana P.Bratu1&Fred Russell Kramer1。
[5] Li Yan, Li Jinming《Clinical molecular diagnosis in Personalized medicine》People's Health Publisher's in August, 2013.
The content of the invention
The purpose of the present invention is not high enough for existing linear probe sensitivity, especially for some rare, nucleic acid
Low sample is measured, such as dry blood cake sample, the nucleic acid concentration extracted is very low, can not be detected using common Taqman sonde methods
SNP, there is provided false positive rate can be reduced, instruct data for clinical drug use, economy easily real-time PCR method, improve real-time fluorescence PCR
Sensitivity a kind of detection human genome competitive real-time fluorescence PCR SNP probes.
It is described it is a kind of detect human genome competitive real-time fluorescence PCR SNP probes, there is secondary structure, comprising with it is complete
Complementary series and do not possess the fluorescence probe of water-disintegrable semicircular structure and fully-complementary sequence entirely.
It is described that there is secondary structure, the base sequence containing SNP site is placed in fluorescence probe 5 ' and held, and in fluorescence probe
Nearby additional unmatched 3~7 bases completely with probe form it into and possess water-disintegrable semicircular structure at 3 ' ends, 3 ' ends with
3~5 bases of 5 ' end addition complete complementaries, increase the water-disintegrable of probe.
During real-time PCR detection, there is high temperature resistant polymerase 5 prime excision enzyme activity and do not produce nonspecific signals.
The fluorescence probe has reporter group through modifying its 5 ' end, and there is quenching group at 3 ' ends；The reporter group includes
ALEX-350、FAM、VIC、TET、CAL Fluor Gold 540、JOE、HEX、CAL Flour Orange 560、TAMRA、
Cal Fluor Red 590、ROX、CAL Fluor 20Red 610、TEXAS RED、CAL Flour Red 635、Quasar
670th, CY3, CY5, CY5.5 or Quasar 705；The quenching group includes DABCYL, BHQ, ECLIPSE or TAMRA.
The base sequence of the SNP site is located at the end of fluorescence probe 5 ', and fluorescence probe 3 ' holds 3~5 additional bases will
Competed jointly with human genome template, held with fluorescence probe 5 ' and carry out complementation.
The fluorescence probe is in 3 ' additional 3~5 bases in end, 5 ' the end complementations with probe.The fluorescence probe is at 3 ' ends
Additional 3~5 bases, preferably 3 bases；The bases G C% is 50%~70%, preferably 60%.
The length of the fluorescence probe can be 25~35 bases, preferably 31 bases.
The fluorescence probe 3 ' holds additional 3~5 bases and probe not complementary, 3~5 bases preferably 4 bases.
In the present invention, SNP is located at the end of fluorescence probe 5 ', is located at fluorescence probe interphase ratio with SNP, SNP is located at fluorescence
Probe 5 ' holds the probability that can substantially reduce false positive.When SNP designs are among the fluorescence probe, if the end of fluorescence probe 5 ' with
Human genome matches completely, even if without SNP mutation, probe also can discharge fluorescence by archaeal dna polymerase digestion, so as to make
Into the result of false positive.If SNP is located at the end of fluorescence probe 5 ', the end of fluorescence probe 5 ' matches completely with template, then fluorescence probe
Fluorescence is discharged by archaeal dna polymerase digestion, and fluorescence probe 5 ' is held and template Incomplete matching, then the end of fluorescence probe 5 ' can stick up
Rise not with the complementation of human genome template, so as to be cut away by archaeal dna polymerase without discharging fluorescence, can so prevent this vacation
Positive generation.
The base sequence of SNP site is located at the end of fluorescence probe 5 '.Fluorescence probe 3 ' holds 3~5 additional bases will be with people
Body genomic templates compete jointly, are held with fluorescence probe 5 ' and carry out complementation.It is glimmering when human genome template has purpose SNP
The end of light probe 5 ' will not be in connection, and hold and combine with fluorescence probe 3 ', so as to send fluorescence.When human genome template
During in the absence of purpose SNP, the end of fluorescence probe 5 ' will be in connection, is hydrolyzed in the real-time PCR extension stage and discharges fluorescence.
Human genome SNP is detected with this.
The SNP is located at the end of fluorescence probe 5 ', and the base sequence of SNP site is placed in into fluorescence probe 5 ' holds, and can improve spy
The specificity of pin.It can be substantially reduced with being located at the end of fluorescence probe 5 ' positioned at the base sequence of fluorescence probe interphase ratio, SNP site
The probability of false positive.When the base sequence design of SNP site is among fluorescence probe, if the end of fluorescence probe 5 ' and human body
Genome matches completely, even if without the base sequence of SNP site, probe also can discharge fluorescence by archaeal dna polymerase digestion,
So as to cause the result of false positive.If the base sequence of SNP site is located at the end of fluorescence probe 5 ', the end of fluorescence probe 5 ' and template
Matching completely, then fluorescence probe discharges fluorescence by archaeal dna polymerase digestion, and the end of fluorescence probe 5 ' and incomplete of template
Match somebody with somebody, then the end of fluorescence probe 5 ' can tilt not complementary with human genome template, glimmering without discharging so as to be turned down by archaeal dna polymerase
Light, it can so prevent the generation of this false positive.
Additional and 3~5 not complementary bases of probe, preferably 4 bases are held in probe 3 '.Additional sequence is held in probe 3 '
Row, it is also not complementary with templet gene group neither with probes complementary.Therefore, be advantageous to improve the hydrolysis efficiency of probe, improve reaction
The sensitivity of system.
The advantage of the invention is that：Sensitivity and the specificity of human genome SNP detections are greatly improved, reduces false positive
Rate, while economical and convenient again.
Brief description of the drawings
Fig. 1 is competitive real-time fluorescence PCR SNP probe schematic diagrams.In Fig. 1, heavy line is complementary with human genome
Sequence, fine line to be additional with 3~7 bases of probes complementary, fine dotted line for it is additional not with probe itself and genome
3~5 complementary bases.
Fig. 2 is the concentration gradient experimental result picture of embodiment 1Taqman average probes.
Fig. 3 is the concentration gradient experimental result picture of the competitive real-time fluorescence PCR SNP probes of embodiment 1.
Fig. 4 is the negative blank assay result figure of embodiment 2Taqman average probes.
Fig. 5 is the negative blank assay result figure of the competitive real-time fluorescence PCR SNP probes of embodiment 2.
Embodiment
Embodiment 1
Folic acid metabolism genetic test, by carrying out the detection of mthfr gene pleomorphism site, assess folic acid metabolism ability.
Detected in real time with general T aqman probes and competitive real-time fluorescence PCR SNP probes.
The present embodiment is using MTHFR as target gene, design specific primer, general T aqman probes and competitive real-time fluorescence
PCR SNP probes, the primer are Primer 1 and Primer 2, and general T aqman probes are Probe1, competitive glimmering in real time
Light PCR SNP probes are Probe2, and sequence is shown in Table 1.Expanding fragment length is 120bp, carry out real-time PCR remolding sensitivity compared with.
Fig. 1 provides competitive real-time fluorescence PCR SNP probe schematic diagrams.
Table 1
| Title | Sequence |
| Primer1 | 5’CCCAAGGAGGAGCTGCTG 3’ |
| Primer2 | 5’ATCACTCACTTTGTGACC 3’ |
| Primer3 | 5’GAGTGGCCGGGAGTTGG3’ |
| Primer4 | 5’CAGCAGACCCTCAAGAC3’ |
| Probe1 | FAM-5’AAGCAAGTGTCTTTGAAGTCT3’-BHQ1 |
| Probe2 | FAM-5’AAGCAAGTGTCTTTGAAGTCTTCGATTCTT3’-BHQ1 |
| Probe3 | FAM-5’AGGCATACACTGAAGTGAAA3’-BHQ1 |
| Probe4 | FAM-5’CTGAAGTGAAAACTGTGAGTGAGTGTTCAG3’-BHQ1 |
* overstriking font is SNP mutation site, and * underscores are additional sequence.
Reaction system volume is 25 μ l, contains 10 × PCR buffer 2.5 μ l, MgCl23mM, dNTP Mix 0.2mM,
2 0.5 μM of 1 0.5 μM of Primer, Primer, 0.05 μM of 0.05 μM of Probe1, Probe2, artificial-synthetic DNA's mutagenesis template
5 μ l, TAKARA Taq HS 0.2 μ l, ddH2O 9.05μl.PCR programs are 95 DEG C of 3min, and cyclic program is 95 DEG C of 15s, 55 DEG C
30s, 72 DEG C of 30s, totally 35 circulations.FAM fluorescent collectings are in the extension stage.Examined using the real-time fluorescence PCR instrument of ABI 7500
Survey.
Real-time PCR testing result such as Fig. 2 and 3, template gradient are 104~101Copies/ μ l, Fig. 2 visit for general T aqman
The result of pin, Fig. 3 are competitive real-time fluorescence PCR SNP probes.From Fig. 2 and 3, the same terms, general T aqman probes
Sensitivity can only be to 102Copies/ μ l, and the sensitivity of competitive real-time fluorescence PCR SNP probes can be to 101copies/μ
l。
Embodiment 2
Alcohol metabolism genetic test, by carrying out the detection of ALDH2 gene polymorphism sites, assess acetaldehyde metabolic capability.
Detected in real time with general T aqman probes and competitive real-time fluorescence PCR SNP probes.
The present embodiment is using ALDH2 as target gene, design specific primer, general T aqman probes and competitive real-time fluorescence
PCR SNP probes, the primer are Primer 3 and Primer 4, and general T aqman probes are Probe3, competitive glimmering in real time
Light PCR SNP probes are Probe4, and sequence is shown in Table 1.Expanding fragment length is 110bp, carries out real-time PCR specificity (false sun
Property) compare.
Reaction system volume is 25 μ l, contains 10 × PCR buffer 2.5 μ l, MgCl23mM, dNTP Mix 0.2mM,
0.5 μM of 0.5 μM of Primer3, Primer4,0.05 μM of 0.05 μM of P3, P4, template are dd H2O 5 μ l, TAKARA Taq
HS 0.2 μ l, ddH2O 9.05μl.PCR programs are 95 DEG C of 3min, and cyclic program is 95 DEG C of 15s, 55 DEG C of 30s, 72 DEG C of 30s, is total to
35 circulations.FAM fluorescent collectings are in the extension stage.Detected using the real-time fluorescence PCR instrument of ABI 7500.
The following Figure 4 and 5 of real-time PCR testing results, are not added with positive template, only add ddH2O, Fig. 4 are general T aqman probes
Result, Fig. 5 is competitive real-time fluorescence PCR SNP probes.From Figure 4 and 5, the same terms, common Taqman probes,
Between SNP designs within the probe, fluorescence signal is generated, and such issues that competitive real-time fluorescence PCR SNP probes can avoid.
The present invention be characterized in that SNP be located at fluorescence probe 5 ' end, and fluorescence probe 3 ' end design 3~
5 bases, combine 5 ' ends of itself and fluorescence probe, so as to form secondary structure.In addition, hold neighbouring additional 3~7 not 3 '
With the base of probe self-complementary.This special construction is greatly improved water-disintegrable, reduction autofluorescent background signal, so as to improve in real time
PCR sensitivity.Increasing the specificity of probe reduces false positive rate.
It the foregoing is only presently preferred embodiments of the present invention.
Sequence table
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Claims (10)
- A kind of 1. competitive real-time fluorescence PCR SNP probes for detecting human genome, it is characterised in that there is secondary structure, comprising With complementary series and not possessing the fluorescence probe of water-disintegrable semicircular structure and fully-complementary sequence completely.
- A kind of 2. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 1, it is characterised in that institute State with secondary structure, the base sequence containing SNP site is placed in into fluorescence probe 5 ' holds, and outer nearby at the end of fluorescence probe 3 ' Add to form it into probe unmatched 3~7 bases completely and possess water-disintegrable semicircular structure, 3 ' ends have been added with 5 ' ends Complete 3~5 complementary bases.
- A kind of 3. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 1, it is characterised in that During real-time PCR detection, there is high temperature resistant polymerase 5 prime excision enzyme activity and do not produce nonspecific signals.
- A kind of 4. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 1, it is characterised in that institute State fluorescence probe has reporter group through modifying its 5 ' end, and there is quenching group at 3 ' ends.
- A kind of 5. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 4, it is characterised in that institute Stating reporter group includes ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Flour Orange 560、TAMRA、Cal Fluor Red 590、ROX、CAL Fluor 20 Red 610、TEXAS RED、CAL Flour Red 635, Quasar 670, CY3, CY5, CY5.5 or Quasar 705.
- A kind of 6. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 4, it is characterised in that institute Stating quenching group includes DABCYL, BHQ, ECLIPSE or TAMRA.
- A kind of 7. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 2, it is characterised in that institute The base sequence for stating SNP site is located at the end of fluorescence probe 5 ', and fluorescence probe 3 ' holds 3~5 additional bases will be with human body gene Group template competes jointly, is held with fluorescence probe 5 ' and carries out complementation.
- A kind of 8. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 1, it is characterised in that institute Fluorescence probe is stated in 3 ' additional 3~5 bases in end, 5 ' the end complementations with probe；The fluorescence probe holds additional 3~5 3 ' Base, preferably 3 bases；The bases G C% is 50%~70%, preferably 60%.
- A kind of 9. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 1, it is characterised in that institute The length for stating fluorescence probe is 25~35 bases, preferably 31 bases.
- A kind of 10. competitive real-time fluorescence PCR SNP probes for detecting human genome as claimed in claim 1, it is characterised in that The fluorescence probe 3 ' holds additional 3~5 bases and probe not complementary, 3~5 bases preferably 4 bases.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109182529A (en) * | 2018-11-01 | 2019-01-11 | 厦门基科生物科技有限公司 | Detect the specific probe and kit in EGFR gene T790M, C797S and the site L798I |
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| WO2007005649A2 (en) * | 2005-06-30 | 2007-01-11 | Applera Corporation | Proximity probing of target proteins comprising restriction and/or extension field |
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| CN109182529A (en) * | 2018-11-01 | 2019-01-11 | 厦门基科生物科技有限公司 | Detect the specific probe and kit in EGFR gene T790M, C797S and the site L798I |
| CN109182529B (en) * | 2018-11-01 | 2021-09-14 | 厦门基科生物科技有限公司 | Specific probe and kit for detecting T790M, C797S and L798I sites of EGFR gene |
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| CN107400722B (en) | 2020-02-07 |
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