CN107400722B - Competitive real-time fluorescent PCR SNP probe for detecting human genome - Google Patents
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Abstract
A competitive real-time fluorescent PCR SNP probe for detecting human genome relates to a single nucleotide polymorphism probe. A fluorescent probe having a secondary structure comprising a completely non-complementary sequence, a hydrolyzable semi-cyclic structure and a completely complementary sequence, wherein the SNP is located at the 5 ' end of the fluorescent probe, and 3 to 5 bases are designed at the 3 ' end of the fluorescent probe and bound to the 5 ' end of the fluorescent probe to form the secondary structure. In addition, 3 to 7 bases which are not complementary to the probe itself are added near the 3' end. The special structure can greatly improve the hydrolyzability and reduce the fluorescence background signal, thereby improving the sensitivity of real-time PCR, increasing the specificity of the probe and reducing the false positive rate. Greatly improves the sensitivity and specificity of human genome SNP detection, reduces the false positive rate, and is economical, simple and convenient.
Description
Technical Field
The invention relates to a Single Nucleotide Polymorphism (SNP) probe, in particular to a competitive real-time fluorescent PCR SNP probe for detecting human genome.
Background
Single Nucleotide Polymorphism (SNP) mainly refers to DNA sequence Polymorphism caused by variation of a Single Nucleotide at the genome level. It is the most common one of the human heritable variations, accounting for over 90% of all known polymorphisms. SNPs are widely existed in human genome, and the average number of the SNPs is 1 in every 500-1000 base pairs, and the total number of the SNPs can be estimated to be 300 ten thousand or more.
SNPs exhibit polymorphisms that involve only single base variations, which can be caused by single base transitions (transitions) or transversions (transitions), or by base insertions or deletions. However, the latter two cases are not included in the so-called SNP.
Theoretically, SNPs may be both allelic polymorphisms and 3 or 4 allelic polymorphisms, but in practice, the latter two are very rare and almost negligible. Thus, the so-called SNPs are all allelic. The variation may be a transition (C T, G A on its complementary strand) or a transversion (ca, gt, cg, at). The incidence of the transition was always significantly higher than that of several other variations, with the SNP with the transition-type variation accounting for about 2/3, and the incidence of several other variations being similar. The Wang et al study also demonstrated this. The chance of switching is high, probably because cytosine residues on CpG dinucleotides are the most vulnerable sites to mutation in the human genome, most of which are methylated and spontaneously deaminated to form thymine.
Since there is a possibility that any base may be mutated in genomic DNA, SNPs may be present in a gene sequence or in a non-coding sequence other than a gene. Generally, there are fewer SNPs (coding SNPs) located in the coding region because within an exon, the variation rate is only 1/5 of the surrounding sequence. However, it is of great significance in the study of genetic diseases, so that the study of cSNP is more concerned.
At present, common methods for detecting SNP comprise a PCR-direct sequencing method, a PCR-pyrosequencing method, a fluorescent quantitative PCR method, a PCR-gene chip method, a PCR-electrophoresis analysis, a PCR-high resolution melting curve method, an allele specific PCR method, a PCR-restriction fragment length polymorphism method, In Situ Hybridization (ISH) and other methods, and the principle and the advantages and the disadvantages of each method are as follows:
1) PCR-direct sequencing method
Also known as PCR-Sanger sequencing, based on the dideoxyribonucleic acid (ddNTP) end-termination method, according to which the nucleotides start to extend at a certain fixed point and randomly terminate at a certain base, and since each base incorporated is fluorescently labeled, four groups of nucleic acid fragments of different lengths ending with A, T, C, G differing by one base are generated; the base sequence of the nucleic acid to be detected is read after separating these fragments by capillary electrophoresis. Sanger sequencing is a classical method of DNA sequence analysis. This method is considered to be a gold standard for genotyping since it can directly read the sequence of DNA.
The operation process of the PCR-Sanger sequencing method mainly comprises four main steps of PCR amplification and PCR product purification, sequencing reaction, sequencing and result analysis. Negative control and positive quality control substances are required to be set during analysis. The method belongs to qualitative detection, and has the advantages of long sequencing length and capability of finding new variable sites. The main defects are as follows: the sensitivity is not high, and particularly when the somatic mutation of the tumor tissue is detected, a false negative result can occur when the mutation proportion of a target gene in the tissue is lower than 20 percent; has special requirements on reagents and instruments, and is not easy to popularize; complex operation, relatively high cost, slow speed and low flux.
2) PCR-Pyrophosphoric acid sequencing method
The method is an enzyme cascade chemiluminescence reaction in the same reaction system catalyzed by 4 enzymes. A sequencing primer marked by biotin is designed in the experiment, after the primer is annealed with single-stranded template DNA, the polymerization of each dNTP on the primer is coupled with the release of a primary fluorescence signal under the synergistic action of 4 enzymes of DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the aim of determining the DNA sequence in real time is fulfilled by detecting the release and the intensity of fluorescence. The required reagents comprise five major classes of sample processing reagents, nucleic acid amplification reagents, single-stranded template preparation reagents, pyrosequencing reagents and positive quality control materials. The required instruments are a PCR instrument and a pyrosequencer. In order to avoid false positive and false negative results, the use of positive quality control products and reaction reagents should be strictly distinguished, and the occurrence of false positive caused by reagent pollution is prevented.
The method has the main advantages that: the detection sensitivity is high, and quantitative detection can be realized on somatic mutation, methylation and the like; accurate and reliable typing, higher flux and flexible experimental design, and can find new mutation or genetic variation. The main disadvantages are that: has special requirements on reagents and instruments, and is not easy to popularize; the detection sensitivity is limited, and false negative is easy to occur on low-abundance somatic mutation (< 3%) in tumor tissues; sequencing is only 10 bases long and cannot analyze long fragments.
3) Real-time fluorescent PCR method
According to the difference of detection principle, the real-time fluorescence PCR method can be divided into probe method and non-probe method, wherein the probe (Taqman and molecular beacon) specifically hybridized with the target sequence is used for indicating the increase of the amplification product, and the fluorescent dye or specially designed primer is used for indicating the increase of the amplification product. The Taqman probe method simultaneously integrates the technologies of nuclease activity at the 5' end, fluorescence and the like, and uses 4 oligonucleotide chains in the reaction process, wherein two oligonucleotide chains are allele specific probes, and two oligonucleotide chains are PCR primers. The two probes can be respectively complementary with the mutant type template and the wild type template, the two ends of the probes are respectively marked by dyes containing a reporter group and a quenching group, and the reporter group fluorescent dyes of the two probes are different. In SNP detection, the annealing process of PCR amplification leads to the hybridization and combination of the probe and the template, when the primer is extended to the probe, the 5 ' end exonuclease activity of the DNA polymerase cuts the 5 ' end reporter group of the probe from the probe, so that the 5 ' end reporter group is separated from the quencher group, and the corresponding fluorescence is released, and the unpaired probe still remains intact and does not fluoresce. Different allele probes emit different fluorescent signals due to different labeled fluorescent dyes, and the genotype of the sample can be judged by detecting the fluorescent signals.
The real-time fluorescence PCR method has the advantages of high sensitivity, accurate typing, simple and quick operation, easy popularization of the used instruments and easy popularization and use. However, the method has low flux and high probe cost, the detection cost of a single site is related to the sample size, and the smaller the sample size is, the higher the cost is. The method is mainly suitable for typing a small number of sites and a large sample. At present, CFDA has approved PCR-fluorescence detection kits for detecting multiple gene polymorphisms such as CYP2C9, VKORC1 and the like.
4) PCR-Gene chip method
The method uses specific oligonucleotide fragments as probes, the specific oligonucleotide fragments are regularly arranged and fixed on a support, then sample DNA is hybridized with a chip according to the base pairing principle through PCR amplification, fluorescent labeling and other programs, and then a fluorescent signal on the chip is detected and analyzed through a fluorescent detection system, so that the genotype information of an individual is rapidly obtained. The operation process of the gene chip parting method comprises PCR nucleic acid amplification, hybridization, chip scanning and result analysis. The method belongs to qualitative detection when used for DNA genotyping, and the sensitivity is 50 ng/. mu.L. When the gene chip method is used for analysis, negative control and positive quality control substances are required to be arranged. When the gene chip detection kit is used, the reagents are required to be stored before being unsealed according to requirements, the liquid of each component is uniformly oscillated before being used, hybridization and developing operations are carried out under the condition of keeping out of the light, PCR reaction liquid and positioning reference are required to be stored under the condition of keeping out of the light, and when the chip is used, the liquid is required to be spread over the whole reaction area, but the liquid cannot overflow and bubbles cannot appear, so that cross contamination is prevented. The method has the main advantage that the base sequences of a plurality of SNP sites to be detected can be detected simultaneously. In China, CFDA has approved a plurality of gene chip kits for polymorphism detection of drug metabolizing enzymes and drug action target genes, such as ALDH2, CYP2C9, CY2C19, CYP2D6, ADR1, ACE and VKORC 1.
5) PCR-electrophoresis analysis
The method is that the target gene segment to be analyzed is amplified by PCR, and the gene polymorphism locus is subjected to genotyping according to the size of a PCR product through agarose gel electrophoresis or capillary electrophoresis analysis. The method belongs to qualitative detection, can only be used for detecting known polymorphic sites, and cannot identify unknown polymorphisms. The agarose electrophoresis method is suitable for detecting the insertion deletion polymorphism with longer fragments, such as ACE insertion deletion polymorphism; capillary electrophoresis is suitable for detecting short indel polymorphisms such as UGT1a1 × 28 polymorphism and microsatellite instability (MSI). In the PCR process, a positive quality control substance and a negative quality control substance need to be established, and the size of the fragment needs to be judged by using a molecular weight marker simultaneously during electrophoretic analysis. When the molecular weight marker reaction tube has no band or a weak band, possible causes include leakage of the spot wells, insufficient or failed fluorescent dye, excessively long electrophoresis time, or excessively high voltage. The method has the advantages of low cost and capability of being developed in a common laboratory; the disadvantage is that it is only suitable for qualitative determination of DNA insertion/deletion polymorphism or fusion gene, and cannot be used for SNP detection.
6) PCR-high resolution melting curve (HRM) method
The method performs genotyping by melt curve analysis of PCR reactions. The melting curve of PCR amplification depends on the amplified sequence, and the difference of one base in the sequence can cause the melting temperature of double-stranded DNA to change. The HRM method uses a real-time fluorescent quantitative PCR instrument to monitor this subtle temperature change and determine whether there is a mutation in the amplified target fragment for genotyping. The HRM analysis uses saturated fluorescent dyes such as LC Green and the like, and the dyes have no inhibition effect on PCR reaction at saturated concentration, so that the dyes can be used at high concentration and are completely combined with minor grooves in a DNA double-helix structure. The rearrangement of fluorescent molecules does not exist in the denaturation process of the double-stranded DNA, and the specificity of the double-stranded DNA is greatly improved, so that the slight change of a melting curve can reflect the difference of bases in amplified fragments. The application of the method to genotyping belongs to qualitative analysis.
The method has the advantages of simple and convenient operation, rapidness, large flux, low use cost and accurate result, is favorable for realizing closed tube operation, and can determine the methylation degree according to a melting curve during methylation detection. The disadvantages of this method are: the inability to exclude newly emerging genetic variations in the nucleic acid to be detected; since the single base mutation causes very little change in the melting temperature of DNA, the method has high requirements on the sensitivity and resolution of the instrument.
7) Allele-specific PCR (AS-PCR)
Also known as Amplification-arresting Mutation system PCR (Amplification Mutation System PCR, ARMS-PCR). The basic principle of the technology is as follows: as Taq DNA polymerase lacks the activity of exonuclease from 3 'end to 5' end, the mismatched base at the 3 'end can cause the extension speed of the primer to become slow, when the mismatch reaches a certain degree, the extension of the primer is stopped, and a PCR amplification product with a specific length can not be obtained, thereby prompting that the template DNA does not have the base matched with the 3' end of the primer, otherwise, the primer has the base. Thus, the AS-PCR reaction requires two allele-specific primers and a common reverse primer, and two non-specific primers mismatch the template at the 3' end, but have identical base sequences in other parts. PCR amplification can only be performed if the 3' end of the primer is fully matched to the template. The PCR product can be analyzed and genotyped by gel electrophoresis. The method can also be combined with real-time fluorescent quantitative PCR for genotyping. The method can be used for detecting various types of SNP, has the advantages of high sensitivity and is particularly suitable for detecting somatic mutation in tumor tissues; the disadvantage is the high false positive rate.
8) PCR-restriction fragment length polymorphism method
Restriction Fragment Length Polymorphism (RFLP) is a method based on the enzyme digestion principle, is one of the earliest classical methods for genotyping, and is still widely adopted at present. The method is mainly based on the principle that certain restriction enzymes can specifically recognize a certain specific sequence and structural DNA and cut the specific sequence and structural DNA. Restriction enzymes generally recognize a specific sequence of double-stranded DNA and cleave the double-stranded DNA at or near a specific position, thereby generating a shorter DNA fragment. Due to the stringency of the recognition sequence of the restriction endonuclease, a change of one base can result in the disappearance of the cleavage activity. By utilizing the characteristic, if the base sequence of the SNP site to be typed is on the recognition site of a certain restriction enzyme, the enzyme has enzyme cutting activity on only one allele. Therefore, when typing SNPs located at restriction enzyme recognition sites, PCR products containing the sites can be incubated with the corresponding restriction enzymes. And (4) carrying out electrophoresis on products after enzyme digestion, and carrying out genotyping according to the sizes of the fragments of the enzyme digestion products. The method does not need any probe or special instrument and equipment, and has the advantages of low cost, simple experimental process and strong operability. The disadvantages are also evident, mainly the throughput is too low, the workload is high for large scale typing and only applies to partial SNP typing.
9) In Situ Hybridization (ISH) method
The ISH method detects abnormalities in a target gene by using various human specimens, including cytological and histological specimens (formalin-fixed paraffin-embedded) prepared by corresponding experimental methods, as targets and performing molecular hybridization using a target DNA probe. The ISH technique can be classified into bright field in situ hybridization (brightfield in situ hybridization) and Fluorescence In Situ Hybridization (FISH) according to the type of the probe label. The target detected by the ISH method has complete cell nucleus, and the extraction of nucleic acid is not needed. The specific methodology is described in the In Situ Hybridization (ISH) guide. In the detection of drug metabolizing enzyme and target gene, the ISH method is mainly used for determining gene amplification and gene deletion abnormality.
Reference documents:
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[3] wangmuijing, Sundaku, Wangling, and the like, the TaqMan probe real-time fluorescent PCR technology is used for detecting 2073A/G single nucleotide polymorphism (J) of AKAP10 gene, proceedings of Sichuan university (medical edition), 2009,40(2), 275-.
[4]Multicolor molecular beacons for allele discriminationSanjayTyagi1,*,Diana P.Bratu1&FredRussell Kramer1。
[5] Lie yan, lie jinming (clinical molecular diagnostics in personalized medicine) national institutes of health press 2013, 8 months.
Disclosure of Invention
The invention aims to provide a competitive real-time fluorescent PCR SNP probe for detecting human genome, which can reduce false positive rate, guide clinical safe medication, is an economical and convenient real-time PCR method and improve the sensitivity of real-time fluorescent PCR, aiming at the problems that the existing linear probe is not high enough in sensitivity, especially for some rare samples with low nucleic acid quantity, such as dry blood spot samples, the concentration of extracted nucleic acid is very low, and SNP cannot be detected by adopting a common Taqman probe method.
The competitive real-time fluorescent PCR SNP probe for detecting the human genome has a secondary structure, and comprises a fluorescent probe which has a completely non-complementary sequence, a hydrolytic semi-ring structure and a completely complementary sequence.
The fluorescent probe with the secondary structure is characterized in that a base sequence containing an SNP site is arranged at the 5 'end of the fluorescent probe, 3-7 bases which are not matched with the probe completely are added near the 3' end of the fluorescent probe to form a semi-ring structure with hydrolyzability, and 3-5 bases which are completely complementary are added at the 3 'end and the 5' end to increase the hydrolyzability of the probe.
In the real-time fluorescent PCR detection process, the kit has high-temperature-resistant polymerase exonuclease activity and does not generate non-specific signals.
The 5 'end of the modified fluorescent probe is provided with a report group, and the 3' end of the modified fluorescent probe is provided with a quenching group; the reporter group comprises 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, Quasar670, CY3, CY5, CY5.5 or Quasar 705; the quencher group comprises DABCYL, BHQ, ECLIPSE or TAMRA.
The base sequence of the SNP locus is located at the 5 ' end of the fluorescent probe, and 3-5 bases added at the 3 ' end of the fluorescent probe compete with the human genome template together and are complementary with the 5 ' end of the fluorescent probe.
The fluorescent probe is added with 3-5 bases at the 3 'end and is complementary with the 5' end of the probe. The 3' end of the fluorescent probe is additionally provided with 3-5 bases, preferably 3 bases; the GC% of the base is 50-70%, preferably 60%.
The length of the fluorescent probe can be 25-35 bases, and 31 bases are preferred.
And 3-5 bases are additionally added at the 3' end of the fluorescent probe and are not complementary with the probe, and 4 bases are preferably selected from 3-5 bases.
In the invention, the SNP is positioned at the 5 'end of the fluorescent probe, and compared with the SNP positioned in the middle of the fluorescent probe, the SNP positioned at the 5' end of the fluorescent probe can greatly reduce the probability of false positive. When the SNP design is located in the middle of the fluorescent probe, if the 5' end of the fluorescent probe is completely matched with the human genome, even if the SNP mutation does not exist, the probe can be cut by DNA polymerase enzyme to release fluorescence, so that a false positive result is caused. If the SNP is located at the 5 'end of the fluorescent probe, the 5' end of the fluorescent probe is completely matched with the template, the fluorescent probe is cut by the DNA polymerase to release fluorescence, and the 5 'end of the fluorescent probe is incompletely matched with the template, the 5' end of the fluorescent probe can be tilted and is not complementary with the human genome template, so that the fluorescent probe is cut by the DNA polymerase to release no fluorescence, and the occurrence of false positive can be avoided.
The base sequence of the SNP site is positioned at the 5' end of the fluorescent probe. The 3-5 bases added at the 3 'end of the fluorescent probe compete with the human genome template together and are complementary with the 5' end of the fluorescent probe. When the SNP of interest exists in the human genome template, the fluorescent probe is not bound to the 5 'end of the human genome template, but is bound to the 3' end of the human genome template, so that the fluorescent probe cannot emit fluorescence. When the target SNP does not exist in the human genome template, the 5' end of the fluorescent probe is combined with the template and is hydrolyzed to release fluorescence in the extension stage of real-time PCR. Thereby detecting human genome SNP.
The SNP is positioned at the 5 'end of the fluorescent probe, and the base sequence of the SNP site is positioned at the 5' end of the fluorescent probe, so that the specificity of the probe can be improved. Compared with the base sequence positioned in the middle of the fluorescent probe, the base sequence of the SNP locus positioned at the 5' end of the fluorescent probe can greatly reduce the probability of false positive. When the base sequence of the SNP site is designed to be positioned in the middle of the fluorescent probe, if the 5' end of the fluorescent probe is completely matched with the human genome, even if the base sequence of the SNP site does not exist, the probe can be cut by DNA polymerase enzyme to release fluorescence, thereby causing a false positive result. If the base sequence of the SNP site is located at the 5 'end of the fluorescent probe, the 5' end of the fluorescent probe is completely matched with the template, the fluorescent probe is cut by the DNA polymerase to release fluorescence, and the 5 'end of the fluorescent probe is incompletely matched with the template, the 5' end of the fluorescent probe can be tilted and is not complementary with the human genome template, so that the fluorescent probe is pushed away by the DNA polymerase without releasing fluorescence, and the occurrence of false positive can be avoided.
3-5 bases, preferably 4 bases, which are not complementary to the probe are added to the 3' end of the probe. The sequence added to the 3' end of the probe is not complementary to the probe, nor to the template genome. Therefore, the hydrolysis efficiency of the probe is improved, and the sensitivity of the reaction system is improved.
The invention has the advantages that: greatly improves the sensitivity and specificity of human genome SNP detection, reduces the false positive rate, and is economical, simple and convenient.
Drawings
FIG. 1 is a schematic diagram of a competitive real-time fluorescent PCR SNP probe. In FIG. 1, the thick solid line represents a sequence complementary to the human genome, the thin solid line represents 3 to 7 bases complementary to the probe, and the thin dotted line represents 3 to 5 bases not complementary to the probe itself and the genome.
FIG. 2 is a graph showing the results of concentration gradient experiments using the Taqman probe of example 1.
FIG. 3 is a graph showing the results of the concentration gradient experiment of the competitive real-time fluorescent PCR SNP probe of example 1.
FIG. 4 is a graph showing the result of a negative blank experiment using the Taqman probe of example 2.
FIG. 5 is a graph of the negative blank test results of the competitive real-time fluorescent PCR SNP probe of example 2.
Detailed Description
Example 1
And (3) detecting the folate metabolism gene, and evaluating the folate metabolism capability by detecting the polymorphic sites of the MTHFR gene. The real-time detection is carried out by using a common Taqman probe and a competitive real-time fluorescent PCR SNP probe.
In the embodiment, MTHFR is used as a target gene, a specific Primer, a common Taqman Probe and a competitive real-time fluorescent PCR SNP Probe are designed, the used primers are Primer1 and Primer 2, the common Taqman Probe is Probe1, the competitive real-time fluorescent PCR SNP Probe is Probe2, and the sequences are shown in Table 1. The amplified fragment length is 120bp, and the sensitivity comparison of real-time PCR is carried out. FIG. 1 shows a schematic diagram of a competitive real-time fluorescent PCR SNP probe.
TABLE 1
Bold font is SNP mutation site, underlined is the additional sequence.
The volume of the reaction system was 25. mu.l, containing 10 XPCR buffer 2.5. mu.l, MgCl23mM, dNTP Mix 0.2mM, Primer 10.5. mu.M, Primer 20.5. mu.M, Probe 10.05. mu.M, Probe 20.05. mu.M, artificially synthesized DNA mutation template 5. mu.l, TAKARA Taq HS 0.2. mu.l, ddH2O9.05. mu.l. The PCR program was 95 ℃ for 3min, and the cycle program was 95 ℃ for 15s, 55 ℃ for 30s, and 72 ℃ for 30s for 35 cycles. FAM fluorescence collection in extended orderAnd (4) section. Detection was performed using an ABI 7500 real-time fluorescent PCR instrument.
The real-time PCR results are shown in FIGS. 2 and 3, and the template gradient is 104～101copies/. mu.l, FIG. 2 shows the results of the common Taqman probe, and FIG. 3 shows the competitive real-time fluorescent PCR SNP probe. As can be seen from FIGS. 2 and 3, under the same conditions, the sensitivity of the ordinary Taqman probe can only be up to 102copies/. mu.l, while the sensitivity of competitive real-time fluorescent PCR SNP probes can reach 101copies/μl。
Example 2
And (3) alcohol metabolism gene detection, namely detecting the polymorphic site of the ALDH2 gene to evaluate the acetaldehyde metabolism capability. The real-time detection is carried out by using a common Taqman probe and a competitive real-time fluorescent PCR SNP probe.
In the embodiment, ALDH2 is used as a target gene, a specific Primer, a common Taqman Probe and a competitive real-time fluorescent PCR SNP Probe are designed, the used primers are Primer3 and Primer4, the common Taqman Probe is Probe3, and the competitive real-time fluorescent PCR SNP Probe is Probe4, and the sequences are shown in Table 1. The amplified fragment was 110bp in length and was subjected to real-time PCR for specificity (false positive) comparison.
The volume of the reaction system was 25. mu.l, containing 10 XPCR buffer 2.5. mu.l, MgCl23mM, dNTP Mix 0.2mM, Primer 30.5. mu.M, Primer 40.5. mu.M, P30.05. mu.M, P40.05. mu.M, template dd H2O 5μl，TAKARA Taq HS0.2μl，ddH2O9.05. mu.l. The PCR program was 95 ℃ for 3min, and the cycle program was 95 ℃ for 15s, 55 ℃ for 30s, and 72 ℃ for 30s for 35 cycles. FAM fluorescence was collected during the extension phase. Detection was performed using an ABI 7500 real-time fluorescent PCR instrument.
The real-time PCR results are shown in FIGS. 4 and 5, in which only ddH was added without adding positive template2O, FIG. 4 shows the results of the regular Taqman probe, and FIG. 5 shows the competitive real-time fluorescent PCR SNP probe. As can be seen from FIGS. 4 and 5, under the same conditions, a common Taqman probe, in which SNP is designed in the middle of the probe, generates a fluorescent signal, and a competitive real-time fluorescent PCR SNP probe can avoid such problems.
The invention is characterized in that the SNP is positioned at the 5 ' end of the fluorescent probe, and 3-5 basic groups are designed at the 3 ' end of the fluorescent probe, so that the SNP is combined with the 5 ' end of the fluorescent probe to form a secondary structure. In addition, 3 to 7 bases which are not complementary to the probe itself are added near the 3' end. The special structure can greatly improve the hydrolyzability and reduce the fluorescence background signal, thereby improving the sensitivity of real-time PCR. Increasing the specificity of the probe decreases the false positive rate.
The foregoing is only a preferred embodiment of the invention.
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Claims (11)
1. A competitive real-time fluorescent PCR SNP probe for detecting human genome is characterized in that the probe has a secondary structure and comprises a fluorescent probe which has a completely non-complementary sequence, a hydrolytic semi-ring structure and a completely complementary sequence;
the fluorescent probe with the secondary structure is characterized in that a base sequence containing an SNP site is placed at the 5 'end of the fluorescent probe, 3-7 bases which are not matched with the probe completely are added near the 3' end of the fluorescent probe to form a semi-ring structure with hydrolyzability, and 3-5 bases which are completely complementary are added at the 3 'end and the 5' end.
2. The competitive real-time fluorescent PCR SNP probe for detecting human genomes of claim 1, which has high temperature resistant polymerase exonuclease activity and does not generate non-specific signals during the real-time fluorescent PCR detection process.
3. The competitive real-time fluorescent PCR SNP probe for detecting human genomes of claim 1, wherein the fluorescent probe is modified to have a reporter group at the 5 'end and a quencher group at the 3' end.
4. The competitive real-time fluorescent PCR SNP probe for detecting the human genome of claim 3, wherein the reporter group comprises ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL flourOrange 560, TAMRA, Cal Fluor Red 590, ROX, CAL Fluor 20Red 610, TEXAS RED, CALUR Red 635, Quasar670, CY3, CY5, CY5.5 or Quasar 705.
5. The competitive real-time fluorescent PCR SNP probe for detecting the human genome of claim 3, wherein the quencher group comprises DABCYL, BHQ, ECLIPSE or TAMRA.
6. The competitive real-time fluorescent PCR SNP probe for detecting human genomes of claim 1, wherein the base sequence of the SNP locus is located at the 5 ' end of the fluorescent probe, and 3-5 additional bases at the 3 ' end of the fluorescent probe compete with the human genome template together and are complementary with the 5 ' end of the fluorescent probe.
7. The competitive real-time fluorescent PCR SNP probe for detecting human genomes of claim 1, wherein the fluorescent probe is complementary to the 5 'end of the probe with the addition of 3-5 bases at the 3' end; the 3' end of the fluorescent probe is additionally provided with 3-5 bases; the GC% of the base is 50-70%.
8. The competitive real-time fluorescent PCR SNP probe for detecting human genomes of claim 1, wherein the length of the fluorescent probe is 25 to 35 bases.
9. The competitive real-time fluorescent PCR SNP probe for detecting the human genome of claim 8, wherein the length of the fluorescent probe is 31 bases.
10. The competitive real-time fluorescent PCR SNP probe for detecting human genomes of claim 1, wherein the 3' end of the fluorescent probe is additionally provided with 3-5 basic groups which are not complementary with the probe.
11. The competitive real-time fluorescent PCR SNP probe for detecting human genomes of claim 10, wherein the 3' end of the fluorescent probe is not complementary to the probe except for 4 bases.
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| CN102586456A (en) * | 2012-03-14 | 2012-07-18 | 上海翼和应用生物技术有限公司 | Method for detecting copy number variations through multiple competitive polymerase chain reaction (PCR) |
| CN102978280A (en) * | 2012-11-01 | 2013-03-20 | 上海翼和应用生物技术有限公司 | Method for detecting copy number variation based on PCR-LDR technology |
| CN103946398A (en) * | 2011-09-15 | 2014-07-23 | 戴维·A·谢弗 | Probe: antiprobe compositions for high specificity dna or rna detection |
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| CN103946398A (en) * | 2011-09-15 | 2014-07-23 | 戴维·A·谢弗 | Probe: antiprobe compositions for high specificity dna or rna detection |
| CN102586456A (en) * | 2012-03-14 | 2012-07-18 | 上海翼和应用生物技术有限公司 | Method for detecting copy number variations through multiple competitive polymerase chain reaction (PCR) |
| CN102978280A (en) * | 2012-11-01 | 2013-03-20 | 上海翼和应用生物技术有限公司 | Method for detecting copy number variation based on PCR-LDR technology |
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| 分子信标-TaqMan 探针法实时荧光定量PCR 新型探针及引物;姜文灿等;《生物技术通报》;20161126;107-114 * |
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Denomination of invention: A competitive real-time fluorescent PCR SNP probe for detecting human genome Granted publication date: 20200207 Pledgee: Bank of China Limited Xiamen Haicang sub branch Pledgor: XIAMEN WIZ BIOTECH CO.,LTD. Registration number: Y2024980011373 |