WO2010135917A1 - 一种检测核酸序列变异的方法 - Google Patents

一种检测核酸序列变异的方法 Download PDF

Info

Publication number
WO2010135917A1
WO2010135917A1 PCT/CN2010/000753 CN2010000753W WO2010135917A1 WO 2010135917 A1 WO2010135917 A1 WO 2010135917A1 CN 2010000753 W CN2010000753 W CN 2010000753W WO 2010135917 A1 WO2010135917 A1 WO 2010135917A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
nucleic acid
amplification
quenching
target
Prior art date
Application number
PCT/CN2010/000753
Other languages
English (en)
French (fr)
Inventor
李庆阁
黄秋英
Original Assignee
厦门大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=43222138&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2010135917(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by 厦门大学 filed Critical 厦门大学
Priority to EP10779999.1A priority Critical patent/EP2436777B1/en
Priority to US13/322,716 priority patent/US8691504B2/en
Priority to CN201080023112.9A priority patent/CN102449167B/zh
Publication of WO2010135917A1 publication Critical patent/WO2010135917A1/zh
Priority to US14/182,067 priority patent/US9334533B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a method for detecting nucleic acid sequence variation and probes and kits therefor, and more particularly to a method for detecting nucleic acid sequence variation by self-quenching probe melting curve, self-quenching probe and reagent box. Background technique
  • the melting curve analysis used to detect nucleic acid sequence variations is to add a temperature rising step (sometimes a cooling step) to the real-time PCR program to detect amplification products or sequence variation information by recording changes in fluorescence with temperature.
  • the current melting point analysis includes three types, namely fluorescent dye method, fluorescent probe method and fluorescent dye combined with fluorescent probe method.
  • the principle of the fluorescent dye method is very simple, that is, a dye that can fluoresce in combination with a double-stranded DNA molecule, such as SYBRGreen, SYT0-9, LC Green, etc., is added to the PCR system, and the temperature rise causes denaturation of the double-stranded DNA to cause a decrease in fluorescence. Melting point changes can indicate sequence changes (Wittwer CT, et al, BioTechniques, 1997 : 22: 130-138; Ririe ⁇ ., et al, Anal. Biochem, 1997, 245: 154-160; US Patent, US 2006/ 0019253 Al); US Patent, US 2003/0224434 Al).
  • changes in single nucleotides can be detected in conjunction with high resolution melting (HRM) analysis (Wittwer C. ⁇ , et al, Clin Chem, 2003, 49: 853-860).
  • HRM high resolution melting
  • the fluorescent probe method uses a probe to detect sequence variation at a specific position, provided that the probe hybridizes with the target sequence to generate a specific fluorescent signal.
  • These probes are various in real-time PCR, but are used for melting curve analysis.
  • FRET probe also known as LightCycler probe, adjacent probe
  • FRET probe also known as LightCycler probe, adjacent probe
  • other uses include single-labeled oligonucleotide probes (US Patent, US 6, 635, 427 B2), HyBeacon (USA) Patent, US 2008/ 0311579 Al) probes, etc.
  • Fluorescent dyes combined with fluorescent probe methods are methods for simultaneously adding fluorescence-sensitizing or fluorescence-quenching dyes and fluorescent probes, such as the so-called induced fluorescence energy resonance transfer iFRET technique (US Patent, US 7, 179, 589 B2), which is Fluorescent intercalation of the dye with the addition of a single-labeled fluorescent probe.
  • Fluorescence-embedded dye-bound double-stranded DNA can increase the fluorescence of the fluorescently labeled probe by energy transfer. The temperature rises to make the probe detach from the target sequence and sensitize. The fluorescence is lowered.
  • the dye method uses a single fluorescence channel detection, and is currently mainly used for the determination of amplification products, combined with HRM for detection of random mutations in amplified sequences, rather than a specific site mutation.
  • the detection is not used for the detection of multiple specific site mutations.
  • Fluorescent dyes combined with fluorescent probe methods, whether they are sensitizing or quenching, are limited to some special fluorescent dyes.
  • the number of channels that can be used to detect fluorescence is limited, and the number of detected sites is also limited. There are very few examples.
  • the LightCyc ler probe consists of two specific probes that are complementary to and adjacent to the template, one labeled with a donor fluorophore called a detection probe and the other labeled with a receptor fluorophore, called an anchor probe, and The melting point of the detection probe is about 10 ⁇ lower than that of the anchor probe, and FRET can occur between the donor fluorophore and the acceptor fluorophore.
  • the two probes are in a free state, and the acceptor fluorophore cannot be excited, so it cannot be detected.
  • the FRET signal is detected; in the presence of a complementary target sequence, the two probes are simultaneously bound to the complementary template, the donor fluorophore and the acceptor fluorophore are close to each other, and the fluorescent energy generated by the excitation fluorophore is excited
  • the FRET signal can be detected by absorption of a fluorescent acceptor group to produce a fluorescent signal of a specific wavelength.
  • the detection probe first dissociates from the template and a specific melting point can be detected.
  • the degree of variability affects the temperature of the dissociation, forming different melting points, and accordingly, it can be judged whether the sequence variation and the specific type of variation occur.
  • the LightCyc er probe uses a method of detecting FRET, a fluorescence donor and a receptor with a suitable combination of wavelengths are required to perform fluorescence energy resonance transfer, and a fluorescent donor and a fluorescent donor capable of performing effective fluorescence energy resonance transfer are currently available. The combination of the body is limited.
  • the optical channel for detecting FRET is different from the conventional method for detecting a single fluorescent dye. Except for specialized instruments, mainstream real-time PCR instruments cannot be used, and the number of FRET detection channels is limited. , making FRET technology very limited in the application of single-tube detection of multiple genetic variants,
  • the single-labeled oligonucleotide probe and the HyBeacon probe in the probe method are both oligonucleotide probes that label only the fluorophore, and the fluorescence intensity changes are generated before and after the probe hybridizes with the target. Both probes can be analyzed for melting curves, and changes in the melting point of the probe are used to detect variations in the nucleic acid sequence.
  • the quenching group is not labeled, and the probe is quenched by a specific nucleic acid sequence or a guanine residue, which has a high fluorescence background, a limited change in fluorescence intensity, and a low letterhead.
  • HyBeacon probe fluorophore is labeled in the probe, which brings some difficulties to the synthesis and labeling of the probe, which limits the probe melting curve method widely used in the detection of nucleic acid sequence variation.
  • a double-labeled probe containing a small groove binder (MGB) in the probe method especially a probe with a MGB located at the 5' end.
  • MGB small groove binder
  • Pleiades probe (Lukhtanov, EA, et al, Nucleic Acids Res, 2007, 35: e30) because it can resist the 5'-hydrolysis activity of heat-resistant DM polymerase (Taq), Also reported for melting curve analysis.
  • the MGB in this type of probe can act to increase the melting point.
  • the purpose of the design is to shorten the probe while maintaining a relatively high melting point. For mismatched target sequences, the melting point is much lower, so it is mainly used for specificity.
  • Detecting matching target sequences is not used for melting curve analysis for mutation detection, because the latter requires that the target sequences, whether matched or mutated, need to be distinguished by different melting points, and do not require mismatched target sequences.
  • the melting point is too low, and the synthesis of such probes is also more difficult and expensive than probes without MGG.
  • fluorescent probes are preferably labeled for common fluorescent groups and can be subjected to multicolor analysis on a universal real-time PCR instrument.
  • Such probes are also preferably suitable for melting curve analysis of nucleic acid amplification products, such as can be conventional
  • PCR cycle reaction conditions are not degraded or only slightly degraded in order to retain enough intact fluorescent probes for subsequent melting curve analysis. More preferably, such a probe must be easily synthesized without involving complicated and expensive chemical modifications, so that the cost of use can be reduced. Summary of the invention
  • the invention provides a method of detecting a variation in a target nucleic acid sequence by performing a melting curve analysis using a self-quenching probe.
  • the probe is labeled with a fluorophore and a quenching group, and can be melted after nucleic acid amplification under the reaction conditions provided by the present invention. Line analysis to detect mutations in the target sequence.
  • the methods provided by the present invention include the use of self-quenching probes and corresponding experimental conditions for melting curve analysis using self-quenching probes.
  • the self-quenching probe generally refers to an oligonucleotide probe having a fluorescent group at one end of the probe and a quenching group at the other end.
  • the fluorescent fluorescence intensity increases when the probe hybridizes to the target nucleic acid sequence.
  • a fluorescent group can be labeled at the 5' end of the probe and a quenching group can be labeled at the 3' end, or a fluorescent group can be labeled at the 3' end of the probe and a quenching group can be labeled at the 5' end. .
  • the fluorophore and the quenching group are close to each other and interact such that fluorescence emitted by the fluorophore is absorbed by the quenching group to weaken probe fluorescence. And when the probe hybridizes to its target nucleic acid sequence, the fluorophore is separated from the quenching group such that fluorescence emitted by the fluorophore is not absorbed by the quenching group and the probe is fluorescent. Enhanced.
  • the sequence of the probe used in the present invention comprises the sequence: a fully complementary sequence of a wild-type or variant target nucleic acid sequence, or a number (e.g., 1 - 10, 1- compared to the fully complementary sequence of a wild-type or variant target nucleic acid sequence) 5, 1-4, 1-3, 1-2, 1 or 2) mismatches, for example one or more (eg 1 - 10, 1-5, 1-4, 1-3, 1-2, 1 or 2) single base conversion, transversion, insertion and/or deletion sequences.
  • a fully complementary sequence of a wild-type or variant target nucleic acid sequence or a number (e.g., 1 - 10, 1- compared to the fully complementary sequence of a wild-type or variant target nucleic acid sequence) 5, 1-4, 1-3, 1-2, 1 or 2) mismatches, for example one or more (eg 1 - 10, 1-5, 1-4, 1-3, 1-2, 1 or 2) single base conversion, transversion, insertion and/or deletion sequences.
  • sequence of the probe used in the present invention may be completely or contain a complementary sequence to its target sequence, or may have one or more single bases of conversion, transversion, insertion compared to the fully complementary sequence of the target nucleic acid sequence. Or a missing sequence.
  • the melting curve analysis generally includes, after the nucleic acid is amplified, the probe that binds to the target sequence, detaches from the target sequence during the temperature increase and causes a change in the fluorescence intensity, and detects the fluorescence intensity with temperature in real time by detecting the process.
  • the change is obtained by obtaining a melting curve of the fluorescence intensity as the ordinate and a temperature as the melting curve of the abscissa, and the melting curve can be used to detect the variation of the target sequence.
  • the above melting curve analysis can also be pressed Another way to cool down is to detect changes in fluorescence from high to low temperatures. Melting curve analysis was performed by data processing.
  • PCR amplification uses heat-resistant nucleic acid without exo-activity or low exo-activity.
  • the probe itself has a chemical modification that is resistant to the exonuclease activity of DNA polymerase.
  • the probe adopts a hairpin structure, which can be either a natural hairpin structure probe or an artificial hairpin structure, but most of them are artificial hairpin structure probes, that is, by artificially adding a target sequence at the end of the probe. Artificial hairpin structure.
  • the rule for adding such a target sequence-independent base is that some or all of the base sequences in the formed hairpin structure are complementary to the target column, and the formed arm length is generally preferably 2 to 15 bases, preferably Between 3 and 7 bases, more preferably between 4 and 7 or 4-6 bases.
  • Another object of the present invention is to provide a method for homogeneously detecting nucleic acid sequence variation, which is achieved by melting curve analysis, a melting curve using a probe method, and the probe used in the present invention is linear or has a hair
  • the probe of the sandwich structure is completely complementary or partially complementary to the target sequence to be analyzed, and the probe is respectively labeled with a fluorescent group and a quenching group, and the probe is characterized by an increase in fluorescence after binding to the target sequence, the probe It is called a self-quenching probe, which means that the fluorescence is relatively weak when the probe is alone, but the fluorescence is increased after hybridization with the sputum sequence.
  • the melting curve analysis and its technical scheme are to design and prepare the corresponding self-quenching probe in the region where the variation of the nucleic acid sequence needs to be detected, and expand
  • the self-quenching probe melting curve analysis after completion of the addition reaction determines whether the nucleic acid sequence of the target region is mutated according to the change of the melting point of the self-quenching probe.
  • Another object of the present invention is to provide a method for simultaneously detecting nucleic acid sequence variations in a plurality of different regions, the technical proposal of which is to design and prepare respective self-quenching probes for each region, and label each self-quenching probe.
  • the invention provides a self-quenching nucleic acid probe for detecting a variation in a target nucleic acid sequence, preferably for detecting a variation in a target nucleic acid sequence by dissolution profile analysis.
  • the probe labels a fluorophore and a quenching group such that the fluorescence (or fluorescence intensity) increases when the probe hybridizes to the target nucleic acid sequence as compared to the absence of the target nucleic acid sequence.
  • the probe has exonuclease activity of DNA polymerase resistant to modifications of 0
  • Another aspect of the invention provides a self-quenching probe, wherein:
  • a fluorophore may be labeled at the 5' end of the probe and a quencher at the 3' end, or a fluorophore may be labeled at the 3' end of the probe and a quencher at the 5' end. ;
  • the fluorophore and the quenching group are close to each other and interact such that fluorescence emitted by the fluorophore is absorbed by the quenching group to weaken probe fluorescence. And when the probe hybridizes to its target nucleic acid sequence, the fluorophore is separated from the quenching group such that fluorescence emitted by the fluorophore is not absorbed by the quenching group and the probe is fluorescent.
  • the probe hybridizes to its target nucleic acid sequence
  • the sequence of the probe may be a fully complementary sequence of its target nucleic acid sequence, or may have one or more than the fully complementary sequence of the target nucleic acid sequence (eg, Sequences such as 1 - 10, 1-5, 1-4, 1-3, 1-2, 1 or 2) single base transformations, transversions, insertions or deletions.
  • Another object of the present invention is to provide a kit for detecting nucleic acid variation using a self-quenching probe-dependent melting curve analysis, the kit comprising one or more of the following components: primers for amplification of the target sequence, self-quenching
  • the probe, and optionally other essential components of the nucleic acid amplification reaction include a refractory nucleic acid polymerase, a single nucleotide, a buffer solution, a metal ion, a buffer of a suitable acidity.
  • the invention may generally comprise the following steps:
  • a melting curve analysis is performed to determine whether the nucleic acid sequence to be detected has a variation and a possible variation type based on the difference in the melting point of the self-quenching probe.
  • a method of detecting a variation in a nucleic acid sequence comprises: 1) designing and preparing a corresponding self-quenching probe in a region where the variation of the nucleic acid sequence is required to be detected, and labeling the fluorescent group (or quenching group) at the 5' end of the probe, the 3' end of the probe Label the quenching group (or fluorophore). If necessary, the probe should be chemically modified and structurally modified to facilitate melting curve analysis; 2) use appropriate primers
  • PCR amplification of the fragment containing the region to be detected PCR amplification requires reaction conditions that facilitate melting curve analysis; 3) melting curve analysis after PCR, the nucleic acid sequence to be detected is determined based on the difference in melting point of the self-quenching probe Whether there is variation and the type of possible variation.
  • the nucleic acid sequence variation refers to a change in a base, which may be a single base change, or a change of two or more bases, including base conversion, transversion, insertion, and deletion.
  • nucleotides or “bases” are used interchangeably and may or may not be modified.
  • the area in which the variation of the nucleic acid sequence needs to be detected may be one or more.
  • the self-quenching probe is an oligonucleotide probe or a DNA analog probe, and its melting point should not be lower than the melting point of the primer, and the length is generally 10-100 bases, preferably 20-60. Bases.
  • the self-quenching probe may be linearly linear in structure, but may also comprise a secondary structure, especially a hairpin structure, and the hairpin structure may be either a natural hairpin structure probe or an artificial hairpin structure.
  • artificial hairpin structure probes that is, artificial hairpin structures are formed by artificially adding target sequence-independent bases at the probe ends.
  • the rule for adding such a target sequence-independent base is that some or all of the base sequences in the formed hairpin structure are complementary to the ⁇ column, and the formed arm length is generally preferably 2-15 bases, preferably Between 3 and 7 bases, more preferably between 4 and 7 or 4-6 bases.
  • the fluorophore and the quenching group are close to each other and interact such that fluorescence emitted by the fluorophore is absorbed by the quenching group to weaken probe fluorescence. And when the probe hybridizes to its target nucleic acid sequence, the fluorophore is separated from the quenching group such that fluorescence emitted by the fluorophore is not absorbed by the quenching group and the probe is fluorescent. Enhance
  • the self-quenching probe distinguishes the wild-type target sequence and the variant target sequence by a change in melting point (or melting curve), and the probe can be designed to be completely complementary to the wild-type target sequence, or the mutant target sequence can be completely complementary.
  • individual mismatched bases can also be introduced in the probe sequence.
  • the self-quenching probe labels a fluorescent group (or a quenching group) at the 5' end and a quenching group (or a fluorescent group) at the 3' end, so that the probe does not hybridize to the target sequence.
  • the interaction between the fluorophore and the quenching group causes the fluorescence emitted by the fluorophore to be absorbed by the quenching group, so that the fluorescence of the probe itself is weak; the probe can form a double-stranded structure when hybridized with the target sequence, When the fluorophore and the quenching group are separated, the fluorescence emitted by the fluorophore cannot be absorbed by the quenching group, and thus the fluorescence of the probe after hybridization increases.
  • the fluorophores include various current fluorescent labels such as ALEX-350, FAM VIC, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL F luor Red 590, R0X, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5. 5, Quasar 705, etc.
  • various current fluorescent labels such as ALEX-350, FAM VIC, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL F luor Red 590, R0X, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5. 5, Quasar 705, etc.
  • the quenching group includes various current quenchers such as DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA.
  • DABCYL DABCYL
  • BHQ e.g., BHQ-1 or BHQ-2
  • ECLIPSE ECLIPSE
  • TAMRA TAMRA
  • the self-quenching probes generally consist of ordinary bases, but may also contain specially modified bases. These specially modified bases can help regulate the binding ability of the probe, such as enhancing probe binding ability or weakening binding ability, and increasing the flexibility of melting curve analysis.
  • a special modified base capable of enhancing the binding ability of the probe such as a locked nucleic acid (ie, locked nuc le ic ac ids, abbreviated as LNA) base, etc., can weaken the binding ability of the universal binding base I and the like.
  • the probes of the invention may be composed of unmodified bases.
  • the base of the probe is modified.
  • the probes of the invention comprise bases that increase or decrease the ability of the probe to bind.
  • the base capable of enhancing probe binding ability comprises a locked nucleic acid base.
  • the base capable of attenuating probe binding ability comprises a universal binding base I.
  • the self-quenching probe can be subjected to PCR amplification using a DNA polymerase having 5' ⁇ 3' exonuclease activity, and the probe can be subjected to 5' ⁇ 3 against DNA polymerase.
  • probes can perform 3' ⁇ 5 f exonuclease activity against DNA polymerase when PCR amplification is performed using DNA polymerase with 3' ⁇ 5' exonuclease activity Modification.
  • the integrity of the probe is maintained throughout the amplification reaction, and subsequent hybridization reactions and melting curve analyses can occur.
  • the modification capable of resisting the activity of the DNA polymerase 5' ⁇ V exonuclease is preferably such that the 5' end of the probe is resistant to the 5' ⁇ 3' exonuclease activity of the nucleic acid polymerase, and the modification method This includes the modification of the linkage between the 5'-end bases, the use of modified base derivatives (such as the use of locked nucleic acid LNA) or the addition of chemical functional groups.
  • a preferred way is to modify the linkage between the 5'-end bases, for example using a phosphorothioate linkage, a methylphosphonate linkage, a boranophosphate linkage, a peptide nucleic acid ( Pept ide nuc le ic ac id ) Linkages that are resistant to exonuclease activity.
  • the oxime is modified with a phosphorothioation linkage located between the first base and the second base at the 5' end.
  • the modification capable of resisting the 5' ⁇ 'exonuclease activity of the DNA polymerase is preferably such that the 3' end of the probe is resistant to the 3' ⁇ 5' exonuclease activity of the nucleic acid polymerase, and the modification includes Modification of the linkage between the 3'-end bases, the use of modified base derivatives (such as the use of locked nucleic acids), or the addition of chemical functional groups.
  • a preferred way is to modify the linkage between the V-terminal bases, e.g., by thiophosphorylation linkages, thiol phosphate linkages, boronic acid phosphorylation linkages, peptide nucleic acid linkages, and the like.
  • a preferred way is to use a phosphorothioation linkage modification, and this modification is located between the first base and the second base at the 3' end.
  • the probe can also be used to facilitate melting curve analysis.
  • the secondary structure preferably a hairpin structure, in particular a hairpin structure in which the probe ends form an arm structure.
  • the manner in which such ends form an arm structure requires, in most cases, artificial addition of target sequence-independent bases at the end of the probe to form an artificial hairpin structure.
  • a certain number of target sequence-independent bases are added to one end or both ends of the probe, so that the ends form an artificial hairpin structure.
  • the rule for adding extraneous bases is that some or all of the arm sequence portions in the hairpin structure are required to be complementary to the array, and the arm length formed is generally preferably 2-15 bases, preferably 3-7 bases. More preferably, it is between 4 - 7 or 4-6 bases.
  • the purpose of this is to ensure that the hybridization between the hairpin structure and the target sequence is sufficiently efficient that it can be effectively used for melting curve analysis.
  • the advantage of using a hairpin probe for melting curve analysis is that, in most cases, the hairpin probe is better resistant to digestion than the linear probe under the same reaction conditions, and the background signal of the hairpin probe is linear. The probe is lower.
  • the amplification conditions favoring the melting curve analysis include an asymmetric amplification mode in which the primer for hybridization of the extension product to the probe is generally between 2 and 100 of the other primer, preferably It is between 2-50 times.
  • said amplification conditions which facilitate melting curve analysis also include those conditions which ensure complete preservation of the probe after amplification, since the probes are pre-added to the reaction tube prior to amplification.
  • the probe itself does not have the ability to resist the 5, exonuclease and exo-enzyme activities of the enzyme, it can be used without the resistance of y-exonuclease and 3'-exonuclease activity.
  • a thermonucleic acid polymerase such as K lentTaq, or a thermostable nucleic acid polymerase with low 5'-exonuclease activity and lacking V-exonuclease activity, such as TaqFS.
  • the probe of the invention may have one nucleic acid segment to be detected, the region comprising an allele nucleic acid sequence to be tested having one or more single nucleotide variations.
  • the probes of the present invention may have two or more nucleic acid segments to be detected, each of the regions comprising an allele nucleic acid to be tested having one or more single nucleotide variations.
  • Sequence, preferably correspondingly self-quenching probes are designed and prepared for each region, and each self-quenching probe is labeled with the same or different fluorophores, and the self-quenching probe after completion of the amplification reaction is melted. Curve analysis, according to the change of the melting point of each self-quenching probe, it is judged whether there is variation in the nucleic acid sequence of the corresponding segment.
  • the number of self-quenching probes of the present invention in a single detection system may be single or multiple.
  • the respective quenching probes can be distinguished from one another by labeling with different fluorescent labeling groups; they can also be labeled by using the same fluorescent labeling group, and Differences in melting points after hybridization of the allele nucleic acid sequences to be tested to achieve mutual discrimination of the respective quenching probes; it is also possible to achieve mutual discrimination of the respective quenching probes by using different fluorescent labeling groups in combination with different melting points.
  • the purpose of increasing the number of detection sections is achieved.
  • the probes of the present invention are generally 5 to 100 bases in length, for example, 10 to 100, 10-50, 15 to 50, 20 to 50, 10 to 40 bases, and further, for example, 10 to 20, 20 - 30, 30 - 40, 15 - 30, 20 - 40, 15 - 25.
  • the basic principles of the invention are as follows:
  • the melting temperature of double-stranded DNA which is also called melting temperature or melting point (Tm).
  • Tm melting temperature
  • the Tm value of double-stranded DNA is fixed.
  • the DNA double strands are completely complementary, the double-stranded structure is relatively stable, and the temperature required for unwinding the DNA double strand is higher, so the Tm value is also higher; when the DNA double strands are not completely complementary, the double-stranded structure formed is less stable, The temperature required to unwind the double strand is low, so the Tm value is also low, and the degree of Tm reduction is also dependent on the specific sequence that is not completely complementary.
  • the probe hybridizes with the target to form a double-stranded structure, and hybridizes with the perfectly matched target, the formed Tm value of the silent chain structure is higher, and the Tm value of the double-stranded structure formed when hybridizing with the target that does not completely match Lower. Therefore, if a change in the Tm value of the probe can be detected, it can be determined whether the target nucleic acid sequence has a variation or even a specific type of variation.
  • Fluorescently labeled probes for nucleic acid sequence variation detection preferably need to meet the following three conditions: First, the probe must have a change in fluorescence intensity before and after hybridization with the target sequence; second, the probe must remain intact during the amplification process, For the melting curve analysis after amplification; third, the probe can not have too strong specificity, otherwise the nucleic acid sequence with mutation is not easy to hybridize with the probe.
  • the self-quenching probe of the present invention can better satisfy the above three conditions. The self-quenching probe hybridizes with the target sequence during the melting curve analysis.
  • the probe forms a rigid, stable double-stranded structure with the target, and the fluorophore is far away from the quenching group, and the fluorophore is emitted.
  • the fluorescence is not absorbed by the quenching group, so a strong fluorescent signal can be detected; as the temperature increases, the probe gradually dissociates from the target, and the probe under dissociation is in a single-strand free-curing state.
  • the needle-labeled fluorophore and the quenching group are in close proximity to each other, and the fluorescence emitted by the fluorophore is absorbed by the quenching group, and only a weak fluorescent signal can be detected at this time.
  • the self-quenching probe melting curve method can be used for the detection of nucleic acid sequence variations. Therefore, according to the present invention, by using a melting curve, the melting point of the hybrid between the probe and the nucleic acid to be tested can be obtained, and based on the melting point, the variation of the nucleic acid to be tested can be detected.
  • a hybrid between the probe and the nucleic acid to be tested and a melting point of the hybrid between the probe and the reference nucleic acid can be obtained, and according to the difference between the two melting points, the test can be detected.
  • the reference nucleic acid can be, for example, a wild type nucleic acid.
  • the same amplification reaction is used to obtain the melting point of the probe and the nucleic acid to be tested and the reference nucleic acid hybrid, or the same melting curve is used to determine the melting point of the probe and the nucleic acid to be tested and the reference nucleic acid hybrid.
  • the same amplification reaction comprises at least one probe, at least one reference nucleic acid, and a plurality of nucleic acids to be detected, thereby detecting variations in the plurality of nucleic acids.
  • the same amplification reaction comprises a plurality of probes, at least one reference nucleic acid, and a plurality of nucleic acids to be detected, thereby detecting a plurality of variations present in the plurality of nucleic acids to be detected.
  • the plurality of probes label different fluorophores.
  • the plurality of probes may be at least 2, 3, 5, 7, 10, 15 or 20, and at most 10, 15, 20, 30 or 50 or more, for example 2 - 5, 2 - 10, 2 - 20, 5 - 10 or 5 - 20.
  • the plurality of nucleic acids to be tested may be, for example, at least 2, 3, 5, 7, 10, 15, or 20, and at most 10, 20, 50, or 100 or more, for example, 2 - 10, 2 - 20, 2 - 50.
  • the plurality of variations may be, for example, at least 2, 3, 5, 7, 10, 15, 20, 30, 50, or 100, and at most 10, 20, 50, 100, or 200 or more, such as 5 - 10, 5 - 20, 5 - 50, 10 - 50, 10 - 100, or 10 - 200.
  • the present invention has the following outstanding advantages:
  • This technology belongs to the homogeneous detection system. After the PCR amplification is completed, only the tube needs to be carried out. The single melting curve analysis can complete the detection. The whole process can be completed on the same fluorescent PCR instrument, or it can be amplified on a common amplification instrument and then transferred to a fluorescent PCR instrument for melting curve analysis. Therefore, the operation is simple and flexible, and the PCR product is not easily contaminated due to the closed process of the entire process.
  • the technology overcomes the limitation of direct detection of the number of gene mutations by real-time PCR.
  • a pattern such as a TaqMan probe, a molecular beacon, a replacement probe, a scorpion primer, etc. is used to detect a sequence variation, that is, a sequence requires a specific The mode of the detection probe.
  • the present invention provides a method for simultaneously detecting multiple sequence variations in a region covered by it using only one self-quenching probe.
  • This technology is easy to synthesize and purify compared to existing probe technologies (such as FRET probes, single-labeled oligonucleotide probes, HyBeacon probes, etc.) that can be used for melting curve analysis.
  • probe technologies such as FRET probes, single-labeled oligonucleotide probes, HyBeacon probes, etc.
  • the method of end labeling for acupuncture is the most common method of labeling), the fluorescence background is low and the signal is high (the probe is self-quenching), and it is easy to multiplex (using multiple different fluorophore labels, each fluorescent
  • the group corresponds to a probe, which can add more than one probe to the same reaction tube, and has low detection cost (a probe can detect multiple variation sites covered by the probe).
  • Figure 1 is a graph of the melting curve of a self-quenching probe in the presence of different target sequences.
  • the left image shows the fluorescence change of the self-quenching probe as a function of temperature.
  • the temperature and fluorescence intensity of the left image are derived and the negative (-dF/dT) is taken to obtain the right image, ie the melting curve.
  • the long line in the figure indicates the matching target sequence (targe t 1); the solid line indicates the single base mismatch target sequence (target 2); the dotted line indicates the targetless sequence.
  • Figure 2 is a sample of different genotypes detected from a quenching probe.
  • the picture on the left is self-quenching The results of real-time PCR detection of the needle; the right panel shows the results of the melting curve analysis of the self-quenching probe after the end of PCR.
  • the black dotted line in the figure indicates the ⁇ / ⁇ genotype, the solid black line indicates - ⁇ 3 7/ — SEA genotype, the solid gray line indicates - ⁇ 4 2/ — SEA genotype, and the gray dotted line indicates negative control ( Negat ive control ).
  • Figure 3 is a graph showing the melting curve of an LNA-modified self-quenching probe in the presence of different target sequences.
  • FIG 4 Multicolor labeling self-quenching probe melting curve single tube detection of hepatitis B virus lamivudine resistance mutation.
  • the yellow (Ye l low ) channel detects Probe 204 and the orange ( Orange ) channel detects Probe 180.
  • the type of mutation represented by each melting curve in the figure is shown by the icon.
  • Linear self-quenching probes of lengths of 26, 30, 36, and 41 nt, respectively, are called 26-nt probe, 30-nt probe, 36-nt probe, 41-nt probe (see Figure 5 for another In 26nt, 30nt, 36nt, and 41nt each)). They are the results of melting curve analysis for target sequences with different degrees of matching. All probes can be analyzed for melting curves and have the ability to distinguish target sequence variations (specific sequences are not shown).
  • Figure 6 Effect of reaction conditions on PCR-melting curve analysis. Amplified by asymmetric PCR, the high temperature resistant polymerase used, whether it is Taq (extraline) with exonuclease activity or TaqFS (dashed line) with greatly reduced exo-activity, using a three-step method (left) The two-step method (right) gives the results of the melting curve analysis. The gray line indicates the absence of a template. None of the melting curve analysis results were obtained using symmetric PCR amplification (not shown).
  • FIG. 7 Melting curve of a hairpin self-quenching probe with different target sequences.
  • the graph on the left shows the denaturing curve of the fluorescence intensity as a function of temperature.
  • the temperature and fluorescence intensity of the left image are derived and the negative number (-dF/dT) is taken to obtain the right image.
  • the melting curve can give the melting point of the hybridization of the different target sequences to the hairpin type self-quenching probe.
  • the solid line in the figure indicates the matching target sequence, the dotted line indicates a single base mismatch ⁇ sequence, and the gray line indicates no target sequence.
  • Figure 8 Hairpin-type self-quenching probes for PCR amplification and melting curve analysis to detect specimens of different genotypes.
  • the left panel shows the results of real-time PCR detection of the self-quenching probe; the right panel shows the results of the melting curve analysis of the self-quenching probe after the end of PCR.
  • the solid black line in the figure indicates the - ⁇ 3 ⁇ 7 -SEA genotype, the black dotted line indicates Nega t ive contro l , the gray solid line indicates - ⁇ 4 2 - SEA genotype, and the gray dotted line indicates ⁇ / ⁇ genotype.
  • FIG. 9 Two probes with the same fluorescent label simultaneously detect the genotype of both mutations. By melting the melting point difference between the two probes by design, the melting point of the high melting point probe (P1) detection mutant is also higher than that of the low melting point probe ( ⁇ 8). The two do not affect each other, and multiple probes can be used to detect multiple genotypes in one channel.
  • the genotypes represented by the various melting curves in the figure are shown as icons.
  • FIG. 10 Multiple mutations in the ⁇ -globin gene were detected by mixing five different fluorescently labeled hairpin-type self-quenching probes in the same reaction tube.
  • the system utilizes five detection channels of the Rotor-gene 6000 to design five corresponding fluorescent substance-labeled probes to detect the genotypes of multiple mutation sites of the ⁇ -globin gene.
  • the small graphs in the figure show the results of detection in five fluorescent channels, and the results from each fluorescence channel correspond to the results of the corresponding fluorescent probe detection.
  • the genotypes represented by the various melting curves in the figure are shown as icons.
  • EXAMPLES 1 Synthesis of different complementary target nucleic acid sequences to investigate linear self-quenching The ability of the probe melting curve method to detect nucleic acid sequence variations.
  • This example designed a self-quenching probe for the 5' untranslated region of the o-globin gene.
  • the self-quenching probe melting curve method is used to distinguish The ability of different target nucleic acid sequences.
  • the self-quenching probe and target nucleic acid sequences used are:
  • Probe 1 5 ' - FAM-CCTGGTGTTTGTTCCTTCCC-BHQ-3' (SEQ ID NO: 1), the linkage between the first base and the second base at the 5' end is thiophosphorylated.
  • Target 1 5' -GCACCGGGAAGGAA back to AAACACCAGGACGCA-3, (SEQ ID NO: 2)
  • Target 2 5' -GCACCGGGAAGGAA AAACACCAGGACGCA-3 > (SEQ ID NO: 3) wherein the underlined portion of the target nucleic acid sequence is complementary to the probe,
  • the base represented by Target 2 is a mutated base, and the target nucleic acid sequence and probe used are synthesized by Shanghai Shenggong Bioengineering Co., Ltd.
  • reaction solution containing 10 X PCR buffer 2. 5 (no Mg 2+ ), 1. 5 mM MgC l 2 , 5 pmol probe 1, no dry nucleic acid sequence or 10 pmol l of one of the above target nucleic acid sequences .
  • the melting curve of the above mixture was analyzed.
  • the reaction procedure was: 95 denaturation for 1 min, 40 incubation for 2 min, followed by a melting curve of 40 ⁇ to 80 at a heating rate of 1 ⁇ / s tep, and a fluorescence signal of the FAM channel was collected.
  • the experiment was performed on a Rotor-Gene 6000 real-time PCR machine.
  • the change in fluorescence intensity is also not obvious; when a target nucleic acid sequence is present, the self-quenching probe forms a rigid and stable double-stranded structure with the complementary target nucleic acid sequence at low temperatures, allowing the fluorophore and quenching group
  • the separation emits fluorescence, and as the temperature increases, the double-stranded structure gradually melts, and the fluorescence gradually decreases.
  • double-stranded structures having different stability are formed, each having a different melting point.
  • the double-stranded structure formed by the self-quenching probe and the fully complementary target nucleic acid sequence (Target 1 ) is relatively stable, so the melting point is high, and the double-stranded structure formed by the mutated target nucleic acid sequence is less stable, so the melting point is lower.
  • the melting point of the self-quenching probe is 66.97 ⁇ ; when the target nucleic acid sequence added is Target 2 with one base mismatch, the melting point of the self-quenching probe is 60. . Therefore, it is possible to judge which target nucleic acid sequence is added based on the difference in melting point of the self-quenching probe. Therefore, the self-quenching probe melting curve method can be used to detect nucleic acid sequence variations.
  • This example designed a self-quenching probe Probe 1 for the 5' untranslated region of the ex-globin gene (see Example 1), which distinguishes the cx l-globin gene from the cx2-globin gene based on the difference in melting point of the probe.
  • the melting curve analysis of Probe 1 is performed to show that the self-quenching probe melting curve method can be used for genotyping.
  • the primers used are:
  • P1 5'-GCAAGCCCTCACGTAGCGAAGTAGAGGAGTCTGAATCTGGA-3' (SEQ ID No: 4) and P2: 5'-GCAAGCCCTCACGTAGCGAATCCCTCTGGCGATAGTCA-3' (SEQ ID No: 5) ⁇
  • the PCR reaction system was: 25 reaction solution containing 10 x PCR buffer 2.5 ⁇ (no Mg 2+ ), 4.0 mM MgCl 2 , 5 pmol probe 1, 0.2 mM dNTP, 1 U hot-start TaqDM polymerase, 0.1 ⁇ upstream primer Pl, 1 ⁇ downstream primer P2, 0.1 ⁇ Probe 1, 5 Human Genome Template (approximately 50ng) or 5 sterile water (negative control).
  • the specimens used were divided into three types: l, - ⁇ 3 ⁇ 7 - SEA (only ct 2-globin gene) and - o 4 7--- SEA (ex ex 1-globin gene only).
  • the PCR reaction conditions were pre-denaturation at 95 ° C for 5 min, cycle period was 95 ⁇ 15 s, 52 ° C for 20 s, n ° 20 s, a total of 50 cycles, and the fluorescence data of the FAM channel was collected in each cycle of annealing.
  • 95 ⁇ was denatured for 1 min, 35 ⁇ was incubated for 2 min, and then the melting curve was increased from 35 to 80 according to the heating rate of VC / step, and the FAM fluorescence signal was collected. This was performed on a Rotor-gene 6000 real-time PCR machine.
  • the linear self-quenching probe Probe 1 exactly matches the sequence of the 5' untranslated region of the ct2-globin gene, and has a base mismatch with the sequence of the 5' untranslated region of the otl-globin gene. As shown in Figure 2, Probe 1 has a higher melting point (65.13 V) when combined with a perfectly matched sequence and a lower melting point (58.48 ⁇ ) when combined with a sequence with a single base difference. Since the fluorescence intensity of the real-time PCR amplification curve itself is relatively poor in reproducibility, the difference in genotypes lies in the difference in fluorescence intensity of the amplification curve (Fig. 2 left), and therefore, it is difficult to Type to distinguish. The analysis of the melting curve after PCR (Fig.
  • the genotype a/a ct has the o l-globin gene and the oc2-globin gene, so it has two melting point peaks; the genotype - o 3 7 - SEA has only the o 2-globin gene, so only the peak with high melting point; Genotype-oc"/- SSA has only the otl-globin gene, so it has only a low melting point. Therefore, the self-quenching probe dissolution curve method can be used for genotyping, as long as the melting point peak and the melting point are high or low. Different genotypes can be distinguished.Example 3. Self-quenching probe melting curve method for synthesizing different complementary sequences for detecting nucleic acid sequence variations.
  • This example designed a self-quenching probe for the DNA region of the DNA polymerase coding region of hepatitis B virus.
  • the artificially synthesized target nucleic acid sequence was used to examine the ability of the LNA-modified self-quenching probe melting curve method to detect nucleic acid sequence variation. .
  • the self-quenching probe used and the nucleic acid sequence are:
  • Probe 204 5' -TET-TTCAGTTAT0T
  • the probe-framed base is replaced with a corresponding locked nucleic acid LNA, and the linkage between the first base and the second base is thiophosphorylated; the target nucleic acid sequence is underlined and probed
  • the needle is complementary, the base of the target nucleic acid sequence is not matched with the base on the probe, wherein 204 Ml, 204 M2, 204 Ml, 204 M3, 204 M4 are wild-type target nucleic acid sequences having different polymorphisms, 204 VI, 204 V2, 204 V3, 204 V4 are target nucleic acid sequences of 204 amino acids with different polymorphisms from methionine to proline. 204 II, 204 12, 204 13 and 204 1 are 204 amino acids with different polymorphisms. A target nucleic acid sequence that changes from methionine to isoleucine.
  • the reaction solution contained 10 X PCR buffer (containing 25 mM Mg 2+ ) 2.5 ⁇ , 0.2 ⁇ probe, and Q. 4 ⁇ M target nucleic acid sequence.
  • the melting curve was analyzed for the above mixture.
  • the reaction procedure was: 95 ⁇ denaturation for 1 min, 40 incubated for 2 min, followed by I / step heating rate from 45 to 76 ⁇ for melting curve analysis, and Y Y Yellow channel fluorescence signal.
  • the experiment was performed on a Rotor-gene 6000 real-time PCR machine.
  • the probes will form different melting points.
  • the melting point of the probe is 65.88" C, 63.88 C, 63.54, 61.79;
  • the target nucleic acid sequence is a target carrying a mutation Nucleic acid sequences 204 VI, 204 V2, 204 V3, 204 V4, 204 11, 204 12, 20413, 20414, the melting points of the probes are 58.23 ⁇ , 54 ⁇ 8, 53.42, 55.62 ° C, 56.07" C, 52.46 ° C 50.1 "C, 52.53.
  • Example 4 Multicolor Labeling Linear Self-quenching Probe Single Tube Simultaneous Detection of Multiple Different Bits Point mutation.
  • the previous examples show that a self-quenching probe can simultaneously cover adjacent mutation sites for simultaneous detection of multiple mutations. This example is used to illustrate that even if non-adjacent mutations are used, single-tube multiplex detection can be achieved by using self-quenching probes labeled with different colors.
  • This example designed self-quenching probes for the B and C regions of the DNA polymerase coding region of hepatitis B virus, respectively, and labeled each probe with a different fluorophore.
  • the probes used were Probe 204 and Probe 180, the primers were F and R, and the sequences of the primers and probes were:
  • Probe 204 (same as Example 3), Probe 180: 5 '-ROX-CCGTTTCTCATGGCTCAGTTTTAG-BHQ-3' (SEQ ID No. 19), F: 5'-GGAAACTGCACTTGTATTCCCATCCCATC-3' (SEQ ID No. 20), R: 5'-GTTTACAGGAAGTTTCCTAAAACAC-3' (SEQ ID No. 21).
  • the boxed base is replaced with the corresponding LNA, and the linkage between the first base and the second base at the 5 ' end is thiophosphorylation.
  • the PCR reaction system is: 25 ⁇ reaction solution containing 10 X PCR buffer 2. 5
  • the types of plasmid templates used include: 204M+180M; 204M+180L1; 204M+180L2; 204V+180L1; 204 I+180LU wherein 204 M+180M means that the amino acid encoded by codon 204 in the C region of the template is methionine, whereas The amino acid encoded by the codon 180 is methionine; 204M+180L means that the amino acid encoded by codon 204 in the C region of the template is methionine, and the codon at position 180 is TTG and the encoded amino acid is leucine; 204M+180L2 The amino acid encoded by the codon 204 in the C region of the template is methionine, and the codon 180 is codon and the encoded amino acid is leucine; 204V+180L1 refers to the amino acid encoded by codon 204 in the C region of the template.
  • the amino acid encoded by TTG is leucine; 204 I+180L1 means that the amino acid encoded by the codon 204 of the C region of the template is isoleucine, and the codon at position 180 of the B region is TTG and the encoded amino acid is leucine. .
  • the PCR reaction conditions were 95 ⁇ 3 min pre-denaturation, and the cycle period was 95 15 s, 50 20 s, 72 20 s, 40 cycles in total, and the fluorescence data of Yellow and Orange channels were collected in each cycle annealing cycle.
  • 95 ⁇ was denatured for 1 min, kept at 40 ° C for 2 min, then increased from 45 to 80 at a heating rate of 1 / step for melting curve analysis, and the fluorescence signals of Yellow and Orange channels were collected.
  • the experiment was performed at Rotor-gene 6000 Performed on a real-time PCR machine.
  • 26-nt probe 5'-R0X-CCTGATACCGACGAGCAAGCACTGGA-BHQ-3' (SEQ ID No. 22) targe t 1 5' -ATTTCCAGTGCTTGCTCGCCGGTATCAGGCTG-3' (SEQ ID No. 23) target 2 5' -ATTTCCAGTGCTTGCTCGCCGGTATCTGGCTG-3' (SEQ ID No. 24) targe t 3 5' -ATTTCCAGCGCTTGCTCGCCGGTATCAGGCTG-3' (SEQ ID No. 25) target 4 5' -ATTTCCAGTGCTTGCTCGCCAGTATCAGGCTG-3' (SEQ ID No.
  • target 5 5' -ATTTCTAGTGCTTGCTCGCCGGTATCTGGCTG-3' SEQ ID No. 27
  • target 6 5'-ATTTCCAGCGCTTGCTCGCCGGTATCTGGCTG-3'
  • target 7 5'-ATTTCCAGCGCTTGTTCGCCGGTATCAGGCTG-3'
  • target 8 5'-ATTTCTAGCGCTTGCTCGCCGGTATCTGGCTG-3'
  • target 9 5' -ATTTCCAGCGCTTGTTCACCTGTATCAGGTTG-3' (SEQ ID No. 31)
  • Target 2 5' -AACAACAATCACATCTACCGCACCAGAGCGAGCCAGTGCATCACAAATTTCCAG
  • Target 3 5 ' -AACGACGATGACATCTACCGCACCAGAGCGAGCCAGTGCATCACAAATTTCCAG
  • Target 4 5' -AACAACAATCACATCTACCGCACCAGAGCGAGCCAATGCATCACAAATCTCCAG
  • Target 1 5'-AACTCCATCACGATCAAGAAGTAACCGGGAAAGCCCATCTGGTTAATCACATCG AGCTC-3' (SBQ ID No. 49)
  • Target 2 5 ' -AACTCCATCACGATCAAGAAGTATCCGGGAAAGCCCATCTGGTTAATCACATCG
  • AGCTC-3' (SEQ ID No. 50)
  • Target 3 5'-AACTCCATCACGATCAAGAAGTAGCCGGGAAAGCCCATCTGGTTAATTACATCG
  • Target 4 5'-AACTCCATTACGATCAAGAAGTATCCGGGAAAGCCCATCTGGTTAATCACATCG
  • Target 5 5'-AACTCCATCACGACCAAGAAGTATCCGGGAAAGCCCATCTGGTTAATCACATCG
  • AGCTC-3' (SEQ ID No. 53)
  • Target 6 5'-AACTCCATCACGATCAAGAAGTAACCGGGAAAACCCATCTGGTTTATCACATCG
  • AGCTC-3' (SEQ ID No. 54)
  • Target 7 S'-AATTCCATCACGATCAAGAAATAGCCGGGAAAGCCCATCTGGTTGATGACATCG
  • PCR amplification cycle uses two-step and three-step methods.
  • the experimental conditions are as follows: PCR amplification system For: 25 reaction system containing 1 PCR buffer (10 mM Tris-HCl, 50 mM KC1, 5% glycerol, pH 8.6), 3.0 mM MgCl 2 , 200 ⁇ dNTPs, 1.0 U Taq or
  • the PCR amplification procedure was: 95'C 3min pre-denaturation, then 95'C 10 s, 68 ⁇ (1 degree per cycle) 10s, 72. C 20 s, a total of 10 cycles, followed by 95'C 10s, 58 10s, 75. C (three-step method) or 58"C (two-step method) 20s, total 40 cycles.
  • the fluorescence signal of the R0X channel is collected during each cyclic annealing phase. After the end of the PCR reaction, the melting curve analysis was carried out.
  • the melting curve analysis program was: 95 denaturation l min, 45 incubation for 5 min, and then the melting curve was analyzed by increasing the heating rate of l " /s tep from 45 ⁇ to 90 ° C.
  • the amplified fragment is the re gene fragment of Vibrio cholerae
  • the upstream primer is 5, -TGTGCGTTTATCGATGCCGAGCAC-3' (SEQ ID No. 56)
  • the downstream primer is 5'-GCTTTTGGTGTCAAAGCCGC-3' (SEQ ID No. 57)
  • linear from Quenching probe is
  • Self-quenching probes by artificially synthesizing target nucleic acid sequences that are fully complementary to the probe and with point mutations, investigate the ability of the hairpin-type self-quenching probe melting curve to distinguish between different target nucleic acid sequences.
  • the self-quenching probe Probe H 5 r -FAM-cgGGTGTTTGTTCCTTCCCG-BHQl-3 r (SEQ ID No. 59) was used, the underlined part of the arm sequence of the hairpin was indicated, and the lower case letter indicated the artificially added target sequence-independent sequence.
  • the fully complementary target sequence is Target-M:
  • Target-UM 5' -ACCGGGAAGGAACAAACACCAGGACGCAAAAAGCA- CGGGGCTGGGCTG-3/ (SEQ ID No. 60), the target sequence containing the mutation is Target-UM:
  • the melting curve analysis system of the synthetic target sequence and the fluorescent probe is: 25 ⁇ L of the reaction solution containing 1 ⁇ SSP buffer [67 mM Tr i s-HCl pH 8. 0 , 16. 6 mM (NH 4 ) 2 S0 4 , 6. 7 mM EDTA, 0. 085 mg/mL BSA], 2. 0 mM MgCl 2 , 0. 1 ⁇ ⁇ hairpin type self-quenching probe 0. 2 ⁇ ⁇ target sequence Target-M or Target-UM or no target sequence (negative control).
  • the melting curve analysis program is: 95 ⁇ denaturation l min, 35 °C, holding for 2 min, followed by I / s tep heating rate, from increasing to 75: melting curve analysis, collecting fluorescence signal of FAM channel during melting curve .
  • This experiment was performed on a Rotor-Gene 6000 real-time PCR machine (Corbett Research, Australia).
  • the neck ring structure of the hairpin self-quenching probe gradually opens during the temperature change from low temperature to high temperature in the absence of the target sequence, and the detected fluorescence signal follows the temperature.
  • the increase is enhanced, and the temperature corresponding to the point where the fluorescence changes most strongly is the melting point of the self-quenching probe self-dimerization structure of the hairpin type.
  • the hairpin-type self-quenching probe emits strong fluorescence at low temperature; as the temperature increases, the fluorescence intensity slowly decreases, approaching the melting point of the double-stranded hybrid formed by hybridization of the probe with the target.
  • the temperature corresponding to the point where the fluorescence changes most strongly is the melting point of the double-stranded hybrid formed by the probe and the target sequence; when the temperature reaches a higher temperature, the fluorescence intensity does not continue to change.
  • the double-stranded hybrids formed by the hairpin-type self-quenching probes hybridized with different target sequences have different stability and thus have different Tm values, and the difference in the target sequences can be judged from the difference in Tm values.
  • Example 7 The hairpin type in Example 7 was used to self-quench Probe H, and the reaction system and reaction conditions in Example 2 (with Probe Probe H instead of Probe Probe 1) were employed.
  • the experimental results are shown in Fig. 8. Since the fluorescence intensity of the real-time PCR amplification curve itself is relatively poor in reproducibility, the difference in genotypes lies in the difference in fluorescence intensity of the amplification curve (Fig. 8 left). It is difficult to distinguish between various genotypes. The analysis of the melting curve after PCR (Fig. 8 right) can distinguish various genotypes.
  • genotype ⁇ ⁇ / ⁇ ⁇ has ⁇ 1-globin gene and ct 2-globin gene, so it has two melting point peaks; genotype - o 3 7 - SEA only o 2- 2-globin gene, so only high melting point Peak; genotype - ⁇ 4 ⁇ 7 - SEA has only c l-globin gene, so there is only a low melting point peak. Therefore, the self-quenching probe dissolution method can be used for genotyping, and different genotypes can be distinguished according to the presence or absence of the melting point peak and the melting point.
  • the self-quenching hairpin probe P1 is perfectly matched to the wild-type target, so the Tm value of the hybridization with the -28 (A>G) mutant target is low; the self-quenching hairpin probe P8 and IVS- 2-654 (OT The mutant target is perfectly matched, so the Tm value of hybridization with the wild type target is low.
  • the PCR amplification system was: 25yL reaction system containing lxSSP buffer [67 mM Tris-HCl, H 8.0, 16.6 mM (NH 4 ) 2 S0 4 , 6.7 mM EDTA, 0.085 mg/mL BSA], 2.0 mMMgCl 2 , 0.2 mM dNTPs , 1 U Taq (HS) (TAKARA, for hot-start Taq, with 5' ⁇ 3' exonuclease activity), O.
  • the PCR amplification program was: 95 min pre-denaturation, cycle period was 95 15 s, 52 ⁇ 20 s, 72 20 s, a total of 50 cycles, and the fluorescence signal of the corresponding detection channel was collected in each cycle annealing stage. After the end of the PCR reaction, the melting curve analysis was carried out.
  • the melting curve analysis program was: denaturation at 95 °C for 1 min, incubation at 35 °C for 2 min, and then increasing the melting rate from 40*C to 80 ⁇ according to the heating rate of ⁇ /step. And collecting the fluorescent signals of the corresponding detection channels. Both real-time PCR and melting curve analysis were performed on a Rotor-Gene 6000 real-time PCR instrument.
  • Multiple mutations can be detected in a single tube using different fluorescently labeled self-quenching probes.
  • This example describes the detection of ⁇ -globin by five different fluorescently labeled hairpin-type self-quenching probes in the same reaction tube.
  • Multiple mutations ie: FAM-labeled probe P1 detects -28 (A>G), -29 (A>G) mutations, R0X-labeled P2 detects CD17 (A>T), CD15/16 (+G And CD14 eight 5 (+G) three mutations, CAL Fluor Red 635 labeled P3 detection IVS-1-1 (G>T), IVS-1-5 (G>C) and CD26 (G>A) three One mutation, HEX-labeled P4 detected CD41/42 (-TCTT) mutation and CD43 (G>T) mutation, and Quasar 705-labeled P5 detected CD71/72 (+A) and CD71/72 (+T) mutations. Mixing these five probes with two pairs of primer
  • the PCR amplification system is: 25
  • the reaction solution contains l xSSP buffer, 3.0 mM MgCl 2 , 0.2 mM dNTPs, 1 U Taq (HS), 0.1 ⁇ M Fl, 1. ⁇ Rl, 0.2 ⁇ F2, 1.6 ⁇ R2, 0.2 ⁇ Pl, 0 ⁇ 2 ⁇ ⁇ 2, 0.1 ⁇ ⁇ 3, 0.3 ⁇ 4, 0 ⁇ 1 ⁇ 5,5 ⁇ L of human genomic DNA template (about 50 ng).
  • the amplification procedure and melting curve analysis procedure of PCR were the same as in Example 9.
  • Figure 10 shows representative results, and the mutant genotypes covered by each probe were correctly detected. Table 2. Examples of primers and probes used in Example 9 and Example 10

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

一种检测核酸序列变异的方法 技术领域
本发明涉及一种检测核酸序列变异的方法及用于该方法的探 针和试剂盒， 尤其是涉及一种通过自淬灭探针熔解曲线检测核酸 序列变异的方法、 自淬灭探针和试剂盒。 背景技术
用于检测核酸序列变异的熔解曲线分析， 就是在实时 PCR程 序中增加一个升温步骤（有时也可以是降温步骤） ， 通过记录荧 光随着温度的变化， 检测扩增产物或者序列变异信息。 目前的熔 点分析包括了三种类型， 分别是荧光染料法、 荧光探针法和荧光 染料联合荧光探针法。
荧光染料法原理十分简单， 就是在 PCR体系中加入能与双链 DNA分子结合发出荧光的染料， 如 SYBRGreen、 SYT0-9、 LC Green 等， 温度升高使双链 DNA发生变性导致荧光降低， 通过熔点变化 可以指示序列变化（Wittwer C. T.， et al, BioTechniques, 1997: 22: 130-138; Ririe Κ· Μ. , et al, Anal. Biochem, 1997， 245: 154-160; 美国专利， US 2006/0019253 Al); 美国专利， US 2003/0224434 Al)。 尤其是结合高分辨熔解（ HRM )分析（Wittwer C. Τ·， et al, Clin Chem, 2003, 49: 853-860)可以检测单个 核苷酸的变化。 荧光探针法就是使用探针检测特定位置的序列变 异， 前提是探针与靶序列杂交后可以产生特异的荧光信号， 这类 探针在实时 PCR中有很多种， 但是用于熔解曲线分析的探针并不 多， 其中最著名的就是荧光能量共振转移探针（FRET探针， 又称 LightCycler 探针、相邻探针）（美国专利， US 7， 160， 998 B2; 美 国专利， US 6, 472, 156 B1; 美国专利， US 6, 140, 054 )， 其它 使用包括单标记的寡核苷酸探针（美国专利， US 6, 635, 427 B2)、 HyBeacon (美国专利， US 2008/ 0311579 Al)探针等。 荧光染料 联合荧光探针法就是同时加入荧光增敏或者荧光淬灭染料和荧光 探针的方法，比如所谓的诱导荧光能量共振转移 iFRET技术（美国 专利， US 7, 179, 589 B2) , 就是加入荧光嵌入染料的同时再加入 单标记的荧光探针， 荧光嵌入染料结合双链 DNA发出的荧光通过 能量转移的方式可以增加荧光标记探针的荧光， 温度升高使得探 针脱离靶序列， 增敏荧光降低。 Gupta 等人（美国专利， US 2007/0020665 Al)公布了一种丙型肝炎病毒分型的方式， 则是在 PCR 反应中同时加入荧光淬灭染料和荧光标记探针， 杂交后发生 荧光淬灭， 温度升高使得探针脱离靶序列， 淬灭的荧光得以恢复 而会使荧光增加。
上述三种熔解曲线方式中， 染料法系采用单一的荧光通道检 测， 目前主要用于扩增产物的判断， 联合 HRM后用于扩增序列中 随机突变的检测， 而不是某一特定位点突变的检测， 更不用于多 个特定位点突变的检测。 荧光染料联合荧光探针法无论是增敏的 方式还是淬灭的方式， 都仅限于一些特殊的荧光染料， 能够用于 检测荧光的通道数目有限， 检测的位点数同样有限， 鉴于此， 目 前应用的例子极少。
探针法中最成功的例子是 LightCyc ler 探针。 LightCyc ler 探针由两条与模板互补、 且相邻的特异探针组成， 其中一条标 记供体荧光基团称为检测探针， 另外一个标记受体荧光基团， 称 为锚探针， 并且检测探针的熔点比锚探针低 10Ό左右， 供体荧光 基团和受体荧光基团之间可以发生 FRET。 在没有靶序列存在时， 两条探针处于游离状态， 受体荧光基团不能被激发， 因此不能检 测到 FRET信号； 在有互补的靶序列存在时， 两探针同时结合在互 补的模板上， 供体荧光基团和受体荧光基团相互靠近， 激发供体 荧光基团产生的荧光能量被荧光受体基团吸收而产生特定波长的 荧光信号， 就可以检测到 FRET信号。 当温度升高， 检测探针首先 从模板解离， 可以检测到一个特定的熔点。 当检测探针杂交的耙 序列存在序列变异， 变异的程度就影响解离的温度， 形成不同的 熔点，据此， 可以判断是否发生了序列变异以及变异的具体类型。 由于这种 LightCyc ler 技术需要一条锚探针， 而锚探针并不用 于检测序列变异， 因此， 锚探针覆盖的区域会造成检测的盲区， 当序列变异广泛存在时， 锚探针区域的选择就变得困难。 此外， 由于 LightCyc l er 探针采用的是检测 FRET的方式， 需要合适的 波长组合的荧光供体和受体才能进行荧光能量共振转移， 而目前 能进行有效荧光能量共振转移的荧光供体和受体组合较有限， 同 时， 检测 FRET 的光学通道不同于常规的检测单个荧光染料的情 况， 除了专门的仪器外， 主流的实时 PCR仪器都无法使用， 加上 FRET检测通道的数目本身也有限， 因此， 使得 FRET技术在单管 检测多个基因变异的应用上受到很大限制，
探针法中的单标记的寡核苷酸探针和 HyBeacon 探针都是只 标记荧光基团的寡核苷酸探针， 探针与靶杂交前后都会产生荧光 强度的变化。 这两种探针都可以进行熔解曲线分析， 利用探针熔 点的变化来检测核酸序列的变异。 然而采用单标记的方式， 没有 标记淬灭基团， 探针依靠特定的核酸序列或鸟嘌呤残基淬灭， 会 有较高的荧光背景， 荧光强度变化有限， 信躁比较低。 此外， HyBeacon探针荧光基团都标记在探针内， 这给探针的合成和标记 都带来了一定的困难， 限制了该探针熔解曲线法广泛用于核酸序 列变异的检测。 探针法中也有一类含有小沟槽结合物（Minor groove binder , 简称 MGB ) 的双标记探针， 特别是 MGB位于 5， 端的探针， 如
MGB-Ec l ipse探针（Afonina, I. A. , et a l, Biotechniques, 2002，
32 : 940-944, 946-949)和 Pleiades探针 ( Lukhtanov, E. A.， et a l, Nucleic Acids Res, 2007， 35: e30 ) 因为可以抵制耐热 DM 聚合酶（Taq ) 的 5' -水解活性， 也报道可用于熔解曲线分析。 这 类探针中的 MGB可以起到提高熔点的作用， 设计的目的在于缩短 探针的同时还能保持相对高的熔点， 而对于不匹配的靶序列， 熔 点则降低很多， 因此主要用于特异地检测匹配的靶序列， 并不用 于突变检测用的熔解曲线分析， 因为后者要求无论是匹配还是突 变的靶序列， 都需要籍有不同的熔点加以区分， 并不需要不匹配 的靶序列的熔点太低， 而且此类探针的合成相对不带 MGG的探针 而言， 也更加困难和昂贵。
因此， 就需要一种新的荧光探针用于熔解曲线分析以实现多 种变异的单管同时检测。 这种荧光探针优选可以进行常见荧光基 团的标记， 可以在通用的实时 PCR仪器上进行多色分析。 这种探 针还优选适合核酸扩增产物的熔解曲线分析， 比如能在常规的
PCR 循环反应条件中不被降解或只有少量降解， 以便保留足够多 的完整的荧光探针用于后续的熔解曲线分析。 这种探针更优选还 必须容易合成， 不涉及复杂的昂贵的化学修饰， 这样才能降低使 用成本。 发明内容
一方面， 本发明提供一种通过用自淬灭探针进行熔解曲线分 析来检测靶核酸序列变异的方法。 该探针标记了荧光基团和淬灭 基团， 在本发明提供的反应条件下可以在核酸扩增后进行熔解曲 线分析， 以检测靶序列的变异。
本发明提供的方法包括了使用自淬灭探针以及使用自淬灭探 针进行熔解曲线分析的相应实验条件。
所述的自淬灭探针一般指的是， 探针的一端标记荧光基团， 另一端标记淬灭基团的寡核苷酸探针。 该探针和靶核酸序列杂交 时荧光荧光强度增加。可在所述探针的 5'末端标记荧光基团而在 3'末端标记淬灭基团， 或可在所述探针的 3'末端标记荧光基团而 在 5'末端标记淬灭基团。 当所述探针单独存在时， 所述荧光基团 与所述淬灭基团彼此接近而相互作用致所述荧光基团发出的荧光 被所述淬灭基团吸收而使探针荧光减弱， 而当所述探针与其靶核 酸序列杂交时， 所述荧光基团与所述淬灭基团相互分离致所述荧 光基团发出的荧光不能被所述淬灭基团吸收而使探针荧光增强。
本发明所用探针的序列包含如下序列： 野生型或变异靶核酸 序列的完全互补序列， 或， 与野生型或变异靶核酸序列的完全互 补序列相比有若干（例如 1 - 10个， 1-5个， 1-4个， 1-3个， 1-2 个， 1个或 2个）错配， 例如具有一个或多个 （例如 1 - 10个， 1-5个， 1-4个， 1-3个， 1-2个， 1个或 2个）单碱基的转换、 颠换、 插入和 /或缺失的序列。
本发明所用探针的序列可以完全是或者包含有与其靶序列的 互补序列， 或可以是与所述靶核酸序列的完全互补序列相比具有 一个或多个单碱基的转换、 颠换、 插入或缺失的序列。
所述的熔解曲线分析一般可以包括， 在核酸扩增后， 与靶序 列结合的探针， 在温度升高过程中脱离靶序列并引起荧光强度变 化， 通过实时检测这一个过程中荧光强度随温度的变化， 获得荧 光强度变化速率为纵坐标， 温度为横坐标的熔解曲线， 利用该熔 解曲线可以检测靶序列的变异情况。 上述熔解曲线分析也可以按 另一种降温的方式获得，也就是从高温到低温，检测荧光的变化。 通过数据处理进行熔解曲线分析。
所述的进行熔解曲线分析的相应实验条件， 指的是能够使自 淬灭探针实现熔解曲线分析的条件， 优选包括如下的一种或者几 种=
1 )使用不对称 PCR, 即反应过程中， 一种引物相对过量， 它 所延伸产生的链与探针杂交；
2 ) PCR扩增使用没有外切活性或者外切活性很低的耐热核酸 聚合醉 ί
3 )探针本身带有能够抵抗 DNA聚合酶的核酸外切酶活性的化 学修饰 ·' 和 /或
4 )探针采用发夹结构， 既可以是天然发夹结构探针也可以是 人工发夹结构， 但多数是人工发夹结构探针， 即通过在探针末端 人为添加靶序列无关碱基形成人工发夹结构。 添加这种靶序列无 关碱基的规则是， 所形成的发夹结构中的臂序列中有部分或者全 部碱基与靶列互补， 并且形成的臂长一般优选在 2- 15个碱基，优 选 3-7个碱基之间， 更优选的是 4一 7个或 4-6个碱基之间。 本发明的另一目的是提供一种均相检测核酸序列变异的方 法， 该方法通过熔解曲线分析来实现， 熔解曲线釆用探针法， 本 发明所使用的探针是一种线性或具有发夹结构的探针并与待分析 靶序列完全互补或部分互补， 探针两端分别标记荧光基团和淬灭 基团， 该探针的特点是与靶序列结合后荧光增加， 这种探针被称 为自淬灭探针， 也就是说该探针单独存在时荧光相对较弱， 但是 与耙序列杂交后荧光增加。 熔解曲线分析及其技术方案是在需要 检测核酸序列变异的区域设计并制备相应的自淬灭探针， 通过扩 增反应完成后的自淬灭探针熔解曲线分析， 根据自淬灭探针熔点 的变化情况， 判断目的区域的核酸序列是否存在变异。 本发明的另一目的是提供同时检测多个不同区域核酸序列变 异的方法， 其技术方案是针对每个区域分别设计并制备相应的自 淬灭探针， 并对每条自淬灭探针标记不同的荧光基团， 通过扩增 反应完成后的自淬灭探针熔解曲线分析， 根据各个自淬灭探针熔 点的变化情况， 判断对应的区域的核酸序列是否存在变异。 另一方面， 本发明提供一种用于检测靶核酸序列变异 （优选 用于通过溶解曲线分析检测靶核酸序列变异）的自淬灭核酸探针。 该探针标记了荧光基团和淬灭基团， 使得与不存在靶核酸序列的 情况相比， 该探针和靶核酸序列杂交时荧光（或荧光强度）增加。 优选地， 该探针带有能够抵抗 DNA聚合酶的核酸外切酶活性的修 饰0
本发明另一方面提供一种自淬灭探针， 其中：
可在所述探针的 5， 末端标记荧光基团而在 3， 末端标记淬灭 基团， 或可在所述探针的 3， 末端标记荧光基团而在 5， 末端标记 淬灭基团；
当所述探针单独存在时， 所述荧光基团与所述淬灭基团彼此 接近而相互作用致所述荧光基团发出的荧光被所述淬灭基团吸收 而使探针荧光减弱， 而当所述探针与其靶核酸序列杂交时， 所述 荧光基团与所述淬灭基团相互分离致所述荧光基团发出的荧光不 能被所述淬灭基团吸收而使探针荧光增强； 且
所述探针的序列可以是其靶核酸序列的完全互补序列， 或可 以是与所述靶核酸序列的完全互补序列相比具有一个或多个 （例 如 1 - 10个， 1-5个， 1-4个， 1-3个， 1-2个， 1个或 2个）单 碱基的转换、 颠换、 插入或缺失的序列。
本发明的另一个目的是提供了利用自淬灭探针依赖的熔解曲 线分析检测核酸变异的试剂盒， 试剂盒包括如下成分中的一种或 者多种： 靶序列扩增的引物， 自淬灭探针， 以及任选的其它核酸 扩增反应的必须组分， 包括耐高温核酸聚合酶， 单核苷酸， 緩沖 溶液， 金属离子， 合适酸度的緩冲液。 本发明一般可以包括以下步骤：
1 )在需要检测核酸序列变异的区域设计并制备相应的自淬灭 探针；
2 )扩增含有待检测区域的片段；
3 )扩增结束后进行熔解曲线分析，根据自淬灭探针的熔点的 差异来判断待检测核酸序列是否具有变异以及可能的变异类型。
在一个具体方面， 提供一种检测核酸序列变异的方法。 该方 法包括： 1 )在需要检测核酸序列变异的区域设计并制备相应的自 淬灭探针， 在探针的 5' 末端标记荧光基团 （或淬灭基团） ， 探 针的 3' 末端标记淬灭基团 （或荧光基团） ， 必要时， 探针需采 用有利于熔解曲线分析的化学修饰和结构改造； 2 )用合适的引物
PCR扩增含有待检测区域的片段， PCR扩增需采用有利于熔解曲线 分析的反应条件； 3 ) PCR结束后进行熔解曲线分析， 根据自淬灭 探针的熔点的差异来判断待检测核酸序列是否具有变异以及可能 的变异类型。 所说的核酸序列变异指碱基的改变，可以是单个碱基的改变， 也可以是两个或两个以上的碱基的改变， 包括碱基的转换、颠换、 插入和缺失。
如果适当的话， 本文所用 "核苷酸" 或 "碱基" 可互换使用， 可经过修饰或未经修饰。
所说的需要检测核酸序列变异的区域可能是一个或多个。 所说的自淬灭探针是一条寡核苷酸探针或 DNA类似物探针， 其本身的熔点应该不低于引物熔点， 长度一般为 10-100个碱基， 优选的是 20-60个碱基。
所说的自淬灭探针在结构上可以是单链线性的， 但也可以包 含二级结构尤其是发夹结构， 发夹结构既可以是天然发夹结构探 针也可以是人工发夹结构， 但多数是人工发夹结构探针， 即通过 在探针末端人为添加靶序列无关碱基形成人工发夹结构。 添加这 种靶序列无关碱基的规则是， 所形成的发夹结构中的臂序列中有 部分或者全部碱基与耙列互补， 并且形成的臂长一般优选在 2-15 个碱基，优选 3-7个碱基之间， 更优选的是 4 - 7个或 4-6个碱基 之间。
当所述探针单独存在时， 所述荧光基团与所述淬灭基团彼此 接近而相互作用致所述荧光基团发出的荧光被所述淬灭基团吸收 而使探针荧光减弱， 而当所述探针与其靶核酸序列杂交时， 所述 荧光基团与所述淬灭基团相互分离致所述荧光基团发出的荧光不 能被所述淬灭基团吸收而使探针荧光增强
所说的自淬灭探针是通过熔点变化 （或者熔解曲线） 变化区 分野生型靶序列和变异靶序列， 探针可以设计成与野生型靶序列 完全互补， 也可以设计成变异靶序列完全互补。 同时为达到上述 目的， 在探针序列中也可以引入个别错配碱基。 所说的自淬灭探针在 5' 末端标记荧光基团 （或淬灭基团）， 3' 末端标记淬灭基团 （或荧光基团）， 因此， 探针在没有与靶序 列杂交时， 荧光基团和淬灭基团互相作用， 导致荧光基团发出的 荧光被淬灭基团吸收， 因而探针本身的荧光很弱； 探针在与靶序 列杂交时， 能够形成双链结构， 使荧光基团和淬灭基团被分离， 荧光基团发出的荧光不能被淬灭基团吸收， 因而杂交后探针的荧 光增加。
所说的荧光基团包括目前各种荧光标记物，如 ALEX-350, FAM VIC, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560， TAMRA, CAL F luor Red 590, R0X, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5. 5, Quasar 705等。
所说的淬灭基团包括目前各种淬灭剂，如 DABCYL、 BHQ类（如 BHQ-1或者 BHQ-2 ) 、 ECLIPSE, 和 /或 TAMRA等。
所说的自淬灭探针一般由普通碱基组成， 但是其中也可以包 含特殊修饰的碱基。 这些特殊修饰的碱基可以帮助调节探针的结 合能力， 例如增强探针结合能力或者削弱结合能力， 增加熔解曲 线分析的灵活性。 比如能够增强探针结合能力的特殊修饰碱基如 锁定核酸（即 locked nuc le ic ac ids , 缩写为 LNA )碱基等， 能 够削弱结合能力的通用结合碱基 I等。 因此，在一个实施方案中， 本发明的探针可由未经修饰的碱基组成。在一个优选实施方案中， 对探针的碱基进行修饰。 在一个优选实施方案中， 本发明的探针 包含能增强或减弱探针结合能力的碱基。 又在一个优选实施方案 中， 所述能增强探针结合能力的碱基包括锁定核酸碱基。 还在一 个优选实施方案中， 所述能减弱探针结合能力的碱基包括通用结 合碱基 I。 在优选实施方案中， 所说的自淬灭探针在使用具有 5' →3' 核酸外切酶活性的 DNA聚合酶进行 PCR扩增时， 探针可以进行抵 抗 DNA聚合酶的 5' →3' 核酸外切酶活性的修饰；在使用具有 3' →5' 核酸外切活性的 DNA聚合酶进行 PCR扩增时，探针可以进行 抵抗 DNA聚合酶的 3' →5f 核酸外切酶活性的修饰。这样在整个 扩增反应中， 探针的完整性得以保持， 可以发生后续的杂交反应 和熔解曲线分析。
所说的能够抵抗 DNA 聚合酶 5' →V 核酸外切酶活性的修 饰， 优选的方式是使探针的 5' 端能抗核酸聚合酶的 5' →3' 核 酸外切酶活性， 修饰方式包括修饰 5' 端碱基之间的连接， 采用 修饰的碱基衍生物 （如使用锁定核酸 LNA ) 或者是增加化学功能 团等。 一种优选的方式是修饰 5' 端碱基之间的连接， 例如采用 硫代磷酸化 （ phosphorothioate ) 连接， 曱基磷酸键 ( methylphosphonate ) 连接， 棚酸磚酸 (boranophosphate)化连 接， 肽核酸（pept ide nuc le ic ac id ) 连接等抗核酸外切活性的 连接。 优选的方式是釆用硫代磷酸化连接修饰， 这种修饰位于于 5' 端的第一个碱基和第二个碱基之间。
所说能够抵抗 DNA聚合酶 5' → ' 核酸外切酶活性的修饰， 优选的方式是使探针的 3' 端能抗核酸聚合酶的 3' →5' 核酸外 切酶活性， 修饰方式包括修饰 3' 端碱基之间的连接， 釆用修饰 的碱基衍生物 （如使用锁定核酸） ， 或者是增加化学功能团等。 一种优选的方式是修饰 V 端碱基之间的连接， 例如采用硫代磷 酸化连接， 曱基磷酸键连接，硼酸磷酸化连接，肽核酸连接等抗核 酸外切活性的连接。 优选的方式是采用硫代磷酸化连接修饰， 而 且这种修饰位于 3' 端的第一个碱基和第二个碱基之间。
在优选的实施方案中， 探针还可以采用有利于熔解曲线分析 的二级结构， 优选的是采用发夹结构， 尤其是探针末端形成臂结 构的发夹结构。 这种末端形成臂结构的方式， 多数情况下需要在 探针末端人为添加靶序列无关碱基形成人工发夹结构。 具体实现 方式是，在探针的一端或者两端添加一定数目的靶序列无关碱基， 使得两端形成人工发夹结构。 添加无关碱基的规则是， 发夹结构 中的臂序列部分中， 需要有部分或者全部与耙列互补， 而且形成 的臂长一般优选在 2-15个碱基，优选 3-7个碱基之间， 更优选的 是 4 - 7个或 4-6个碱基之间。这样做的目的是保证发夹结构与靶 序列之间杂交有足够高的效率， 使之可以有效用地于熔解曲线分 析。 使用发夹探针进行熔解曲线分析的好处是， 在多数情况下， 同样的反应条件下， 发夹探针比线性探针更好地耐受酶切， 而且 发夹探针的背景信号比线性探针更低。
在优选的实施方案中， 所说的有利于熔解曲线分析的扩增条 件包括釆用不对称扩增模式， 其延伸产物与探针杂交的引物一般 是另一条引物的 2-100之间， 优选的是 2-50倍之间。
在优选的实施方案中， 所说的有利于熔解曲线分析的扩增条 件还包括那些保证扩增后探针得以完整保存的条件， 因为探针都 是在扩增之前预先加入到反应管内。 比如， 在探针本身不具备抵 抗酶的 5， -外切酶和 3， -外切酶活性的能力时， 就可以釆用不具 备 y -外切酶和 3' -外切酶活性的耐热核酸聚合酶，如 K lentTaq, 或者采用 5' -外切酶活性 f艮低并且缺乏 V -外切酶活性的耐热 核酸聚合酶， 如 TaqFS。 具备上述性质的酶很多， 具体实猃都可 以参考上述要求加以选择。
在一个实施方案中， 本发明的探针的待检核酸区段可以是一 个， 所述区域包含具有一种或多种单核苷酸变异的待检等位核酸 序列。 在一个优选实施方案中， 本发明的探针的待检核酸区段可以 是两个或两个以上， 所述各区域各包含具有一种或多种单核苷酸 变异的待检等位核酸序列， 优选针对每个区域分别设计并制备相 应的自淬灭探针， 并对每条自淬灭探针标记相同或不同的荧光基 团， 通过扩增反应完成后的自淬灭探针熔解曲线分析， 根据各个 自淬灭探针熔点的变化情况， 判断对应区段的核酸序列是否存在 变异。
本发明的自淬灭探针在一个检测体系中的数目可以是单个， 也可以是多个。 在使用多个自淬灭探针时， 可通过使用不同的荧 光标记基团进行标记而实现各自淬灭探针的彼此区分； 也可通过 使用相同的荧光标记基团进行标记， 并利用其与待检等位核酸序 列杂交后的熔点差异来实现各自淬灭探针的彼此区分； 也可通过 在使用不同的荧光标记基团的同时结合使用不同的熔点来实现各 自淬灭探针的彼此区分， 从而达到增加检测区段数目的目的。
本发明的探针的长度一般是 5 - 100 个碱基， 例如 10 - 100 个， 10-50个， 15到 50个， 20到 50个， 10 - 40个碱基， 再例如 10 - 20, 20 - 30, 30 - 40, 15 - 30， 20 - 40个， 15 - 25个。 本发明的基本原理如下：
尽管下文描述了本发明假设的原理， 但是， 本发明的范围并 不受到这些原理的限制。
在 DM热变性过程中，有 50%DNA变性解链时的温度称为双链 DNA 的解链温度， 又称熔解解温度或熔点 （Tm ) 。 在溶剂固定的 前提下，双链 DNA的 Tm值是固定不变的。当 DNA双链完全互补时， 形成的双链结构较为稳定， 使 DNA双链解开所需温度较高， 故 Tm 值也较高； DNA 双链不完全互补时， 形成的双链结构较不稳定， 使双链解开所需温度较低， 故 Tm值也较低， 而且 Tm降低的程度 也是依赖不完全互补的具体序列的。
基于上述理论， 探针与靶杂交形成双链结构， 与完全匹配的 靶杂交， 则形成的默链结构的 Tm值较高， 若与不完全匹配的靶杂 交时， 形成的双链结构 Tm值较低。 因此， 若能检测到探针 Tm值 的变化， 就能判断靶核酸序列是否存在变异， 乃至变异的具体类 型。
荧光标记的探针要用于核酸序列变异检测优选需要满足以下 三个条件： 一是探针与靶序列杂交前后必须有荧光强度的变化； 二是探针在扩增过程中必须保持完整， 以用于扩增后的熔解曲线 分析； 三是探针不能有过强的特异性， 否则具有变异的核酸序列 不易与探针杂交。 本发明所述的自淬灭探针则能较好的满足以上 三个条件。 自淬灭探针在熔解曲线分析过程中， 低温阶段与靶序 列杂交， 这时探针与靶形成刚性、 稳定的双链结构， 荧光基团与 淬灭基团距离较远， 荧光基团发出的荧光不能被淬灭基团吸收， 因此可以检测到很强的荧光信号； 随着温度的升高， 探针逐渐从 靶上解离， 解离下的探针呈单链自由卷曲状态， 探针标记的荧光 基团和淬灭基团互相靠近， 荧光基团发出的荧光被淬灭基团所吸 收， 此时只能检测到微弱的荧光信号。 对自淬灭探针在熔解曲线 分析过程中进行荧光信号的检测， 就能观察到探针与靶的杂交和 解离过程， 形成荧光强度随着温度变化而变化的曲线， 即探针的 熔解曲线，对熔解曲线求导分析，就可以找到荧光变化最强的点， 对应的温度即是探针的 Tm值。探针与完全匹配的靶杂交时形成的 双链结构 Tm值最高，与具有不同序列变异的靶杂交时形成的双链 Tm较低， 不同的变异类型则可以形成不同的 Tm值。 因此， 自淬 灭探针熔解曲线法可以用于核酸序列变异的检测。 因此， 根据本发明， 采用熔解曲线， 可以获得探针与待测核 酸之间杂交体的熔点， 根据该熔点， 可以检测待测核酸的变异。
或者， 优选地， 根据本发明， 采用熔解曲线， 可以获得探针 与待测核酸之间杂交体以及探针与参照核酸之间杂交体的熔点， 根据这两个熔点的差异， 可以检测待测核酸的变异。 参照核酸可 以是例如野生型核酸。
优选地， 用同一个扩增反应获得探针与待测核酸和参照核酸 杂交体的熔点, 或者， 用同一个熔解曲线测定反应物获得探针与 待测核酸和参照核酸杂交体的熔点。 更优选地， 同一个扩增反应 中包含至少一个探针、 至少一个参照核酸和多个待测核酸， 由此 检测多个核酸的变异。 更优选地， 同一个扩增反应中包含多个探 针、 至少一个参照核酸和多个待测核酸， 由此检测多个待测核酸 中存在的多个变异。优选地， 所述多个探针标记不同的荧光基团。 所述多个探针可以是至少 2、 3、 5、 7、 10、 15或者 20个， 并且 最多 10个、 15个、 20个、 30或者 50个或者更多， 例如 2 - 5个、 2 - 10个， 2 - 20个、 5 - 10个或者 5 - 20个。 所述多个待测核酸 可是例如至少 2、 3、 5、 7、 10、 15、 或者 20个， 并且最多 10个、 20个、 50个或者 100个或者更多， 例如 2 - 10个、 2 - 20个， 2 - 50个。 所述多个变异可是例如至少 2、 3、 5、 7、 10、 15、 20、 30、 50、 或者个 100， 并且最多 10、 20、 50、 100、 或者 200个或 者更多， 例如 5 - 10个， 5 - 20个， 5 - 50个， 10 - 50个， 10 - 100个， 或者 10 - 200个。 与现有核酸序列变异检测技术相比， 本发明具有以下突出优 点:
1 )该技术属于均相检测系统， PCR扩增完成后只需要进行筒 单的熔解曲线分析就可以完成检测， 整个过程可以无需开盖， 既 可以在同一台荧光 PCR仪上完成， 也可以在普通扩增仪上扩增后 再转移到荧光 PCR仪器进行熔解曲线分析， 因此操作简便、灵活， 而且由于整个过程闭管操作， 不易造成 PCR产物污染。
2 )该技术克服了实时 PCR直接检测基因突变数目的限制， 比 如目前采用 TaqMan探针、 分子信标、 置换探针、 蝎子引物等探针 检测序列变异的模式， 即一种序列需要一种特异的检测探针的模 式。 与此不同的是， 本发明提供的方法是仅用一条自淬灭探针就 可以同时进行其所覆盖区域多种序列变异的检测。
3 ) 该技术与现有的可以用于熔解曲线分析的探针技术（如： FRET探针、 单标记的寡核苷酸探针、 HyBeacon探针等）相比， 具 有易于合成和纯化 （探针釆用末端标记的方式是最普通的标记方 式） 、 荧光本底较低和信躁比较高 （探针能够自我淬灭） 、 易于 多重检测 （釆用多个不同荧光基团标记， 每一个荧光基团对应一 种探针， 可以在同一反应管中加^ 多种探针） 、 检测成本低（一 种探针可以检测其所覆盖的多个变异位点） 等优点。 附图说明 图 1为不同靶序列存在下的自淬灭探针的熔解曲线图。 左图 显示的是自淬灭探针随着温度变化相应的荧光变化情况， 将左图 的温度与荧光强度的变化求导并取其负数 (-dF/dT)就得到右图， 即熔解曲线， 直接反应了在不同靶序列存在情况下自淬灭探针的 熔点。 图中的长线段虛线表示匹配靶序列（targe t 1) ; 实线表示 单碱基错配靶序列（target 2) ; 点状虚线表示无靶序列。
图 2是自淬灭探针检测不同基因型的标本。 左图为自淬灭探 针实时 PCR检测的结果； 右图是 PCR结束后对自淬灭探针熔解曲 线分析结果。 图中黑色虛线表示 αα/αα基因型， 黑色实线表示 -α3 7/— SEA基因型， 灰色实线表示 - α4 2/— SEA基因型， 灰色虛线表示阴 性控制 ( Negat ive control ) 。
图 3为不同靶序列存在下 LNA修饰的自淬灭探针的熔解曲线 图。
图 4 多色标记自淬灭探针熔解曲线法单管检测乙型肝炎病 毒拉米夫定耐药突变。 其中黄色 （Ye l low )通道检测的为 Probe 204 , 橙色 （Orange )通道检测的为 Probe 180。 图中各个熔解曲 线所代表的突变类型如图标所示。
图 5. 线性自淬灭探针长度对熔解曲线的影响。 长度分别是 26、 30、 36、 41nt碱基的线性自淬灭探针，分别称作 26-nt probe, 30-nt probe, 36-nt probe, 41-nt probe (分另 'J见图 5中 26nt, 30nt, 36nt，和 41nt各图） 。 它们对于匹配程度不同的靶序列 ( target ) 的熔解曲线分析结果。 所有探针都可以都可以进行熔 解曲线分析， 并具备区分靶序列变异的能力 （图中未标注具体序 列） 。
图 6. 反应条件对 PCR-熔解曲线分析法的影响。用不对称 PCR 扩增的， 所用的耐高温聚合酶无论是具有外切酶活性的 Taq (实 线）还是外切活性大为降低的 TaqFS (虛线） , 使用三步法 （左 图）还是两步法 （右图） ， 都能给出熔解曲线分析结果。 灰线表 示不存在模板的情况。 而凡是使用对称 PCR扩增都未得到熔解曲 线分析结果（图中未给出） 。
图 7. 发夹型自淬灭探针与不同靶序列的熔解曲线图。 左图 显示的是荧光强度随温度变化相应的变性曲线， 将左图的温度与 荧光强度的变化求导并取其负数（-dF/dT)就得到右图， 即熔解曲 线。 熔解曲线可以给出在不同靶序列与发夹型自淬灭探针杂交的 熔点。 图中实线表示匹配靶序列，虛线表示单个碱基错配靼序列， 灰线表示无靶序列。
图 8. 发夹型自淬灭探针用于 PCR扩增后熔解曲线分析检测 不同基因型的标本。 左图为自淬灭探针实时 PCR检测的结果； 右 图是 PCR结束后对自淬灭探针熔解曲线分析结果。 图中黑色实线 表示 -α3· 7 一 SEA基因型， 黑色虛线表示阴性控制 （ Nega t ive contro l ) , 灰色实线表示 - α4 2—— SEA基因型， 灰色虛线表示 αα/αα 基因型。
图 9. 相同荧光标记的两种探针同时检测两种突变的基因 型。 通过设计将两个探针熔点相差拉大， 高熔点探针（P1 )检测 突变型的熔点也高于低熔点探针（Ρ8 ) 的野生型。 这样二者就互 不影响， 可以用一个通道中使用两个探针检测多个基因型。 图中 各个熔解曲线所代表的基因型如图标所示。
图 10. 在同一反应管内， 用五种不同荧光标记的发夹型自淬 灭探针混合检测 β-珠蛋白基因的多个突变。 该体系利用了 Rotor-gene 6000 的五个检测通道， 设计五种相应荧光物质标记 的探针， 分别检测了 β-珠蛋白基因的多个突变位点的基因型。 图 中各小图给出了在五个荧光通道中检测的结果， 每个荧光通道给 出的结果对应于相应荧光探针检测的结果。 图中各个熔解曲线所 代表的基因型如图标所示。 实施例
以下实施例结合附图对本发明作进一步的说明， 所给出的是 本发明的一些具体实施例， 这些实施例只是说明而不表示本发明 所有的可能性， 本发明并不局限于这些实施例中提到的材料、 反 应条件或参数， 任何在相关领域具备经验的人， 都可以按照本发 明的原理， 利用其它类似材料或反应条件实现本发明所描述的对 突变进行检测。 这些并不脱离本发明描述的基本概念。
实施例 1. 人工合成不同的互补靶核酸序列考察线性自淬灭 探针熔解曲线法用于检测核酸序列变异的能力。
本实例设计了一条针对 o -珠蛋白基因 5' 非翻译区的自淬 灭探针， 通过人工合成与探针完全互补和带有点突变的靶核酸序 列， 考察自淬灭探针熔解曲线法区分不同靶核酸序列的能力。 所 用的自淬灭探针和靶核酸序列为：
Probe 1： 5 ' - FAM-CCTGGTGTTTGTTCCTTCCC-BHQ- 3' (SEQ ID NO: 1) , 5' 端第一个碱基和第二个碱基之间的连接采用硫代磷 酸化连接。
Target 1： 5' -GCACCGGGAAGGAA回 AAACACCAGGACGCA-3， (SEQ ID NO: 2) Target 2 : 5' -GCACCGGGAAGGAA AAACACCAGGACGCA-3> (SEQ ID NO: 3) 其中， 靶核酸序列加下划线部分与探针互补， Target 2加框 表示的碱基为突变的碱基， 所用的靶核酸序列和探针都在上海生 工生物工程有限公司合成。
25 反应液中含 10 X PCR buffer 2. 5 (无 Mg2+)， 1. 5 mM MgC l2， 5 pmol probe 1， 不加乾核酸序列或者加入 10 pmo l 上 述靶核酸序列中的一种。 对上述混合液进行熔解曲线分析， 反应 程序为： 95 变性 1 min， 40 保温 2 min， 随后按 1 Ό / s tep 的升温速率从 40Ό递增至 80 进行熔解曲线分析， 并且采集 FAM 通道荧光信号。 该实验在 Rotor-Gene 6000实时 PCR仪上进行。
结果见图 1， 从图中我们可以观察到， 在没有靶核酸序列存 在时， 自淬灭探针处于单链自由卷曲状态， 荧光基团由与淬灭基 团相互靠近， 荧光基团发出的荧光被淬灭基团所淬灭， 随着温度 的变化， 荧光强度的变化也不明显； 当有靶核酸序列存在时， 自 淬灭探针低温时与互补的靶核酸序列形成刚性且稳定的双链结 构， 使荧光基团和淬灭基团分离而发出荧光， 随着温度的升高， 双链结构逐渐解链， 而荧光逐渐下降。 对不同的靶核酸序列， 形 成稳定性不同的双链结构， 具有各自不同的熔点。 其中自淬灭探 针与完全互补的靶核酸序列 （Target 1 )形成的双链结构较稳定， 故熔点较高， 与突变的靶核酸序列形成的双链结构稳定性较差， 故熔点较低。 当加入的靶核酸序列为 Target 1时， 自淬灭探针的 熔点为 66.97Ό; 当加入的靶核酸序列为有一个碱基不匹配的 Target 2时， 自淬灭探针的熔点为 60. 。 因此， 根据自淬灭 探针熔点的不同就可以判断加入的是哪种靶核酸序列。 因此， 自 淬灭探针熔解曲线法可以用于检测核酸序列变异。 实施例 2. 用线性自淬灭探针 PCR-熔解曲线分析法检测不同 基因型的标本。
本实例设计了针对 ex-珠蛋白基因 5' 非翻译区的自淬灭探 针 Probe 1 (见实施例 1)， 根据探针熔点的差别来区分 cx l-珠蛋 白基因和 cx2-珠蛋白基因， 使用人类基因组模板， 实时 PCR扩增 后， 对 Probe 1进行熔解曲线分析， 来说明自淬灭探针熔解曲线 法可以用于基因分型。 所用的引物为：
P1: 5' -GCAAGCCCTCACGTAGCGAAGTAGAGGAGTCTGAATCTGGA-3' ( SEQ ID No:4 ) 和 P2: 5' -GCAAGCCCTCACGTAGCGAATCCCTCTGGCGATAGTCA-3' ( SEQ ID No: 5 ) β
PCR反应体系为： 25 反应液中含 10 xPCR buffer 2.5 μΐ (无 Mg2+) , 4.0 mM MgCl2， 5 pmol probe 1， 0.2 mM dNTP, 1 U 热 启动 TaqDM聚合酶， 0.1 μΜ上游引物 Pl， 1 μΜ下游引物 P2, 0.1 μΜ Probe 1, 5 人类基因组模板（约 50ng ) 或者 5 的无菌水（阴性控制） 。 所使用的标本分为三种型： l 、 - α 3· 7— SEA (只有 ct 2-珠蛋白基因）和 - o 47--SEA (只有 ex 1-珠蛋白 基因）。 PCR反应条件为 95°C 5 min预变性， 循环周期为 95Π 15 s， 52°C 20s, n° 20 s， 共 50个循环， 在每个循环退火阶段 釆集 FAM通道荧光数据。 PCR反应后， 95Π 变性 1 min, 35Π 保 温 2 min, 随后按 VC / step的升温速率从 35 递增至 80 进行 熔解曲线分析， 并且采集 FAM荧光信号。 该实猃在 Rotor-gene 6000实时 PCR仪上进行。
线性自淬灭探针 Probe 1与 ct2-珠蛋白基因 5' 非翻译区的 序列完全匹配， 而与 otl-珠蛋白基因 5' 非翻译区的序列有一个 碱基不匹配。 如图 2所示， Probe 1与完全匹配的序列结合时熔 点较高（65.13V)， 与有单个碱基差异的序列结合时熔点较低 (58.48Ό)。由于实时 PCR扩增曲线本身的荧光强度高低的重现性 相对较差， 而各基因型的差别就在于扩增曲线的荧光强度差异上 (图 2左图） ， 因此， 很难对各种基因型进行区分。 而 PCR后的 熔解曲线分析 （图 2右图） 则可以很好的区分各种基因型。 基因 型 a/a ct具有 o l-珠蛋白基因和 oc2-珠蛋白基因，故具有两个 熔点峰； 基因型 - o 37— SEA只有 o 2-珠蛋白基因， 故只有高熔点 的峰； 基因型 -oc"/—SSA只有 otl-珠蛋白基因， 故只有低熔点的 峰。 因此， 自淬灭探针溶解曲线法可以用于基因分型， 只要根据 熔点峰的有无及熔点的高低就可以对不同的基因型进行区分。 实施例 3. 人工合成不同的互补序列的自淬灭探针熔解曲线 法用于检测核酸序列变异的能力。
乙肝病毒的 DNA聚合酶编码区 C 区 204位密码子由蛋氨酸 ( methionine, M )发生碱基突变成为纈氨酸 ( val ine, V ) ( ATG →ATT ) 或异亮氨酸（ i soleucine, I ) ( ATG→ GTG ) 就会导致对 一线药物拉米夫定的耐药， 并且很可能伴有第 180位密码子由亮 氨酸（ leuc ine, L ) 突变为蛋氨酸（M ) ( CTG/TTG→ ATG ) 。
本实实例设计了针对乙肝病毒的 DNA聚合酶编码区 C区的自 淬灭探针，用人工合成的靶核酸序列考察 LNA修饰的自淬灭探针 熔解曲线法用于检测核酸序列变异的能力。 所用的自淬灭探针和 把核酸序列为：
Probe 204: 5' -TET-TTCAGTTAT0T |GATGATGTGG-BHQ-3' (SEQ ID No. 6)
204 Wl: 5' -CAAAACCACATCATCCATATAACTGAAAGCCAAA- V (SEQ ID No. 7)
204 W2: 5' -CAAAACCACATCATCCATATAACTAAAAGCCAAA- (SEQ ID No. 8)
204 W3: 5' -CAAAACCACATCATCCATATAGCTGAAAGCCAAA- 3' (SEQ ID No. 9)
204 W4: 5' -CAAAAACACATCATCCATATAACTGAAAGCCAAA- 3' (SEQ ID No. 10)
204 VI: 5' -CAAAACCACATCATCCACATAACTGAAAGCCAAA- 3' (SEQ ID No. 11)
204 V2: 5' -CAAAACCACATCATCCACATAACTAAAAGCCAAA- V (SEQ ID No. 12)
204 V3: 5' -CAAAACCACATCATCCACATAGCTGAAAGCCAAA- 3' (SEQ ID No. 13)
204 V4: 5' -CAAAAACACATCATCCACATAACTGAAAGCCAAA- V (SEQ ID No. 14)
204 11: 5' -CAAAACCACATCATCAATATAACTGAAAGCCAAA- V (SEQ ID No. 15)
204 12: 5' -CAAAACCACATCATCAATATAACTAAAAGCCAAA- V (SEQ ID No. 16)
204 13: 5' -CAAAACCACATCATCAATATAGCTGAAAGCCAAA- 3' (SEQ ID No. 17)
204 14: 5' -CAAAAACACATCATCAATATAACTGAAAGCCAA-3' (SEQ [D No. 18)
其中 ，探针加框的碱基用相应的锁定核酸 LNA替代，并且 端第一个碱基和第二个碱基之间的连接釆用硫代磷酸化连接； 靶 核酸序列加下划线部分与探针互补， 靶核酸序列加粗的碱基与探 针上的碱基不匹配， 其中 204 Ml, 204 M2、 204 Ml , 204 M3、 204 M4为具有不同多态性的野生型靶核酸序列， 204 VI、 204 V2、 204 V3、 204 V4为具有不同多态性的 204位点氨基酸由蛋氨酸变成为 缬氨酸的靶核酸序列， 204 II、 204 12、 204 13、 204 1 为具 有不同多态性的 204位点氨基酸由蛋氨酸变成为异亮氨酸的靶核 酸序列。
上述靶核酸序列和探针都在上海生工生物工程有限公司合 成-
25 反应液中含 10 X PCR buffer (含 25mM Mg2+) 2.5 μΐ, 0.2 μΜ探针， Q.4 μ M的靶核酸序列。 对上述混合液进行熔解 曲线分析， 反应程序为： 95Ό 变性 1 min， 40 保温 2 min, 随后按 I / step的升温速率从 45 递增至 76Ό进行熔解曲线分 析， 并且釆集 Yellow通道荧光信号。 该实验在 Rotor-gene 6000 实时 PCR仪上进行。
结果见图 3， 从图中我们可以观察到， 加入各种的靶核酸序 列， 探针会形成各自不同的熔点。 当靶核酸序列是野生型靶核酸 序列 204 Wl、 204 W2、 204 W3、 204 W4时， 探针的熔点依次是 65.88"C, 63.88。C、 63.54 、 61.79 ; 当靶核酸序列是携带突 变的靶核酸序列 204 VI、 204 V2、 204 V3、 204 V4、 204 11、 204 12、 20413、 20414,探针的熔点依次是 58.23Ό、54· 8 、53.42 、 55.62°C、 56.07"C、 52.46°C、 50.1 "C、 52.53 。 虽然有个别熔 点比较接近，但是 4种野生型和 8种突变型之间熔点区别比较大， 即使是区分最小的野生型（204 W3)和突变型（204 VI), 两者熔点 差异为 3· 56 。 因此， LNA修饰的自淬灭探针熔解曲线法用于检 测核酸序列变异。 实施例 4. 多色标记线性自淬灭探针单管同时检测多个不同 位点的突变。 前面的实施例表明，一个自淬灭探针可以同时覆盖邻近的突 变位点， 进行多个突变同时检测。 本实例则用来说明即使不相邻 的突变， 通过使用不同颜色标记的自淬灭探针， 亦可以实现单管 多重检测。
本实实例设计了分别针对乙肝病毒的 DNA聚合酶编码区 B区 和 C区的自淬灭探针， 并对每个探针标记不同的荧光基团。 所用 的探针为 Probe 204和 Probe 180， 引物为 F和 R， 引物和探针的 序列为：
Probe 204 ( 同 实 施 例 3)， Probe 180: 5 ' -ROX-CCGTTTCTCATGGCTCAGTTTACTAG-BHQ-3' (SEQ ID No. 19) , F: 5' -GGAAACTGCACTTGTATTCCCATCCCATC-3' (SEQ ID No. 20) , R: 5' -GTTTACAGGAAGTTTCCTAAAACAC-3' (SEQ ID No. 21)。
其中的探针部分， 加框的碱基用相应的 LNA替代， 并且 5' 端 第一个碱基和第二个碱基之间的连接采用硫代磷酸化连接。
PCR反应体系为： 25μΙ反应液中含 10 X PCR buffer 2. 5
(无 Mg2+)， 4. 0 mM MgC , 0. 2 mM dNTP, 1 U 热启动 Taq DNA 聚合酶， 0. 1 μ Μ上游引物 F, Ι μ Μ下游引物 R， 0. 2 μ Μ 探针， 5 人工构建质粒模板或无齒水（阴性控制） 。 所用的质粒模板 的类型包括： 204M+180M; 204M+180L1; 204M+180L2; 204V+180L1; 204 I+180LU 其中 204 M+180M指模板的 C区 204位密码子编码 的氨基酸为蛋氨酸，而 Β区 180位密码子编码的氨基酸为蛋氨酸； 204M+180L指模板的 C区 204位密码子编码的氨基酸为蛋氨酸， 而 Β区 180位密码子为 TTG且编码的氨基酸为亮氨酸； 204M+180L2 指模板的 C区 204位密码子编码的氨基酸为蛋氨酸， 而 Β区 180 位密码子为 CTG且编码的氨基酸为亮氨酸； 204V+180L1指模板的 C区 204位密码子编码的氨基酸为缬氨酸， 而 Β区 180位密码子 为 TTG且编码的氨基酸为亮氨酸； 204 I+180L1指模板的 C区 204 位密码子编码的氨基酸为异亮氨酸， 而 B区 180位密码子为 TTG 且编码的氨基酸为亮氨酸。
PCR反应条件为 95Ό 3 min预变性， 循环周期为 95 15s, 50 20s, 72 20s, 共 40个循环， 在每个循环退火阶段釆集 Yellow和 Orange通道荧光数据。 PCR反应后， 95Ό 变性 1 min, 40"C 保温 2 min, 随后按 1 / step的升温速率从 45 递增至 80 进行熔解曲线分析， 并且采集 Yellow和 Orange通道荧光信 号。 该实验在 Rotor- gene 6000实时 PCR仪上进行。
结果见图 4， 探针 Probe 204 (Yellow 通道）和 Probe 180 (Orange通道）都可以根据其熔点对模板的基因型进行区分， 单管 放置多个不同颜色标记的自淬灭探针并不会造成探针之间的干 扰， 每条探针都能够很好的检测各自覆盖区域的突变， 因此， 对 自淬灭探针标记进行多色标记， 就可以同时检测多个不同位点的 突变。 实施例 5. 线性自淬灭探针长度对熔解曲线分析影响的考察
我们设计了长度分别是 26、 30、 36、 41碱基的线性自淬灭探 针， 分另1 J称作 26-nt probe, 30-nt probe, 36-nt probe, 41-nt probe (见表 1)。 考察它们对于匹配程度不同的靶序列（target) 的熔解曲线分析结果。 反应条件如下： 25 反应液中含 2.5 μΐ 10 X PCR buffer [10mM Tr is-HCl, 50mMKCl, 5% glycerol (W/V) , pH 8.6], 3.0 mM MgCl2, 0.2 μ 探针， 不加靶核酸序列或者加 入终浓度为 0.4 μΜ 的上述靶核酸序列中的一种。 对上述混合液 进行熔解曲线分析， 反应程序为： 95 变性 1 min， 保温 5 min, 随后按 1"C/ step的升温速率从 递增至 90 进行熔解 曲线分析。
结果（见图 5 )表明， 这些不同长度从 26、 30、 36、 41碱基 的线性自淬灭探针都可以进行熔解曲线分析， 而且都具备区分靶 序列变异的能力。 四种不同长度的线性自淬灭探针及其靶序列
Probes / Sequences
targe t s
26-nt probe 5' -R0X-CCTGATACCGACGAGCAAGCACTGGA-BHQ-3' (SEQ ID No. 22) targe t 1 5' -ATTTCCAGTGCTTGCTCGCCGGTATCAGGCTG-3' (SEQ ID No. 23) target 2 5' -ATTTCCAGTGCTTGCTCGCCGGTATCTGGCTG-3' (SEQ ID No. 24) targe t 3 5' -ATTTCCAGCGCTTGCTCGCCGGTATCAGGCTG-3' (SEQ ID No. 25) target 4 5' -ATTTCCAGTGCTTGCTCGCCAGTATCAGGCTG-3' (SEQ ID No. 26) target 5 5' -ATTTCTAGTGCTTGCTCGCCGGTATCTGGCTG-3' (SEQ ID No. 27) target 6 5' -ATTTCCAGCGCTTGCTCGCCGGTATCTGGCTG-3' (SEQ ID No. 28) target 7 5' -ATTTCCAGCGCTTGTTCGCCGGTATCAGGCTG-3' (SEQ ID No. 29) target 8 5' -ATTTCTAGCGCTTGCTCGCCGGTATCTGGCTG-3' (SEQ ID No. 30) target 9 5' -ATTTCCAGCGCTTGTTCACCTGTATCAGGTTG-3' (SEQ ID No. 31)
30-nt probe 5' -ROX-CACTGGAAATTTGTGATGCATTGGCTCGCT-BHQ-3' (SEQ ID No. 32) target 1 5' -AACGACAATCACATCTACCGCACCAGAGCGAGCCAATGCATCACAAATTTCCAG
TGC-3' (SEQ ID No. 33)
target 2 5' -AACAACAATCACATCTACCGCACCAGAGCGAGCCAGTGCATCACAAATTTCCAG
TGC-3' (SEQ ID No. 34)
target 3 5 ' -AACGACGATGACATCTACCGCACCAGAGCGAGCCAGTGCATCACAAATTTCCAG
CGC-3' (SEQ ID No. 35)
target 4 5' -AACAACAATCACATCTACCGCACCAGAGCGAGCCAATGCATCACAAATCTCCAG
Figure imgf000028_0001
target 1 5'-AACTCCATCACGATCAAGAAGTAACCGGGAAAGCCCATCTGGTTAATCACATCG AGCTC-3' (SBQ ID No. 49)
target 2 5 ' -AACTCCATCACGATCAAGAAGTATCCGGGAAAGCCCATCTGGTTAATCACATCG
AGCTC-3' (SEQ ID No. 50)
target 3 5'-AACTCCATCACGATCAAGAAGTAGCCGGGAAAGCCCATCTGGTTAATTACATCG
AGCTC-3' (SBQ ID No. 51)
target 4 5'-AACTCCATTACGATCAAGAAGTATCCGGGAAAGCCCATCTGGTTAATCACATCG
AGCTC-3' (SEQ ID No. 52)
target 5 5'-AACTCCATCACGACCAAGAAGTATCCGGGAAAGCCCATCTGGTTAATCACATCG
AGCTC-3' (SEQ ID No. 53)
target 6 5'-AACTCCATCACGATCAAGAAGTAACCGGGAAAACCCATCTGGTTTATCACATCG
AGCTC-3' (SEQ ID No. 54)
target 7 S'-AATTCCATCACGATCAAGAAATAGCCGGGAAAGCCCATCTGGTTGATGACATCG
AGCTC-3' (SEQ ID No. 55) 实施例 6. 反应条件对 PCR-熔解曲线分析法的影响
我们分别比较对称 PCR和不对称 PCR, 使用具有外切活性的 Taq酶和外切酶活性降低的 TaqFS酶， PCR扩增循环釆用两步法和 三步法， 实验条件如下： PCR扩增体系为： 25 反应体系中含 有 1 PCR buffer (10 mM Tris-HCl, 50 mM KC1, 5% glycerol, pH 8.6), 3.0 mM MgCl2, 200 μΜ dNTPs, 1.0 U Taq 或者
TaqFS, 0.04 μΜ 上游引物， 0.4μΜ下游引物， 0. ΙμΜ 探针， 5 的质粒模板（ 1.0χ 105 copies) ， 水作为阴性控制。 PCR扩 增程序程序为： 95'C 3min 预变性，然后是 95'C 10 s， 68Ό (每 一循环降低 1度） 10s， 72。C 20 s, 共 10个循环， 接下来是 95'C 10s, 58 10s, 75。C (三步法）或者 58"C (两步法） 20s， 共计 40个循环。 在每个循环退火阶段釆集 R0X通道的荧光信号。 PCR 反应结束后， 进行熔解曲线分析， 熔解曲线分析程序为： 95 变 性 l min， 45 保温 5 min，随后按 l " /s tep 的升温速率从 45 Π 递增至 90'C 进行熔解曲线分析。扩增的片段是霍乱弧菌的 re 基因片段，上游引物是 5, -TGTGCGTTTATCGATGCCGAGCAC-3' (SEQ ID No. 56)，下游引物是 5' -GCTTTTGGTGTCAAAGCCGC-3' (SEQ ID No. 57)，线性自淬灭探针是
5, -ROX-CCTGATACCGACGAGCAAGCACTGGA-BHQ2-3/ (SEQ ID No. 58)。
实验结果如图 6可以看出， 凡是使用对称 PCR扩增都未得 到熔解曲线分析结果（未列出） ， 用不对称 PCR扩增的， 所用的 耐高温聚合酶无论具有外切酶活性的 Taq还是外切活性大为降低 的 TaqFS, 使用三步法还是两步法， 都能给出熔解曲线分析结果。 虽然在使用 Taq时， 酶切的程度会大些， 但是基本不影响实猃结 果。 实施例 7. 人工合成的互补靶序列考察发夹型自淬灭探针熔解曲 线法用于检测靶序列变异的能力 本实例设计了一条针对 ct -珠蛋白基因 5' 非翻译区的发夹 型自淬灭探针， 通过人工合成与探针完全互补和带有点突变的靶 核酸序列， 考察发夹型自淬灭探针熔解曲线法区分不同靶核酸序 列 的 能 力 。 所 用 的 自 淬 灭 探 针 Probe H: 5r -FAM-cgGGTGTTTGTTCCTTCCCG-BHQl-3r (SEQ ID No. 59)，下划 线表示发夹的臂序列部分， 小写字母表示人工添加的靶序列无关 序列。 完全互补靶序列为 Target-M:
5' -ACCGGGAAGGAACAAACACCAGGACGCAAAAAGCA- CGGGGCTGGGCTG-3/ (SEQ ID No. 60)， 含有突变的靶序列为 Target-UM:
5' -ACCGGGAAGGAAgAAACACCAGGACGCAAAAAGCA- CGGGGCTGGGCTG-3, (SEQ ID No. 61)。 下划线部分与探针互补， 加框字母表示相应的碱基突变所在的位置。
人工合成的靶序列与荧光探针的熔解曲线分析体系为： 25 μ L反应液中含有 1 χ SSP buffer [67 mM Tr i s-HCl pH 8. 0 , 16. 6 mM (NH 4 ) 2 S04， 6. 7 mM EDTA, 0. 085 mg/mL BSA] , 2. 0 mM MgCl 2 , 0. 1 μ Μ 的发夹型自淬灭探针 0. 2 μ Μ 的靶序列 Target-M 或 Target-UM 或不加靶序列 （阴性控制） 。 熔解曲线分析程序为： 95匸变性 l min, 35 °C , 保温 2 min， 随后按 I /s tep升温速率， 从 递增至 75 :进行熔解曲线分析， 在熔解曲线过程中采集 FAM通道的荧光信号。 本实验在 Rotor-Gene 6000 实时 PCR仪 ( Corbet t Research，澳大利亚）上进行。
如图 7 (左图） 所示， 没有靶序列存在时， 由低温到高温的温 度变化过程中， 发夹型自淬灭探针自身的颈环结构逐渐打开， 检 测到的荧光信号随着温度的升高而增强， 其中荧光变化最强的点 对应的温度即为发夹型自淬灭探针自身二聚结构的熔点。 当有互 补的靶序列存在时， 低温时发夹型自淬灭探针发出强烈的荧光； 随着温度的升高， 荧光强度緩慢下降， 接近探针与靶杂交形成的 双链杂交体的熔点时， 荧光强度下降速率加快， 其中荧光变化最 强的点对应的温度即是探针与靶序列形成的双链杂交体的熔点； 到达较高温时， 荧光强度不再继续变化。 发夹型自淬灭探针与不 同的靶序列杂交形成的双链杂交体稳定性不同， 因而具有不同的 Tm值， 从 Tm值的差异就可以判断靶序列的差异。 如图 7 (右图） 所示， 发夹型自淬灭探针自身二聚结构形成的熔点峰与双链杂交 体形成的熔点峰朝向不一致， 二者完全不会干扰， 并且与双链杂 交体形成的熔点峰相比， 探针自身的二聚结构形成的峰几乎可以 忽略不计。 因此， 发夹型自淬灭探针也可以用于熔解曲线分析。 实施例 8. 发夹型自淬灭探针 PCR -熔解曲线分析法检测不同基因 型的标本
釆用实施例 7中的发夹型自淬灭 Probe H , 并且采用实施例 2 中的反应体系和反应条件 （用探针 Probe H代替探针 Probe 1 ) 。 实验结果如图 8， 由于实时 PCR扩增曲线本身的荧光强度高低的 重现性相对较差， 而各基因型的差别就在于扩增曲线的荧光强度 差异上（图 8左图）， 因此， 很难对各种基因型进行区分。 而 PCR 后的熔解曲线分析 （图 8右图） 则可以很好的区分各种基因型。 基因型 α α / α α具有 α 1-珠蛋白基因和 ct 2-珠蛋白基因，故具有 两个熔点峰； 基因型 - o 3 7—SEA只有 o 2-珠蛋白基因， 故只有高 熔点的峰； 基因型 - α 4·7— SEA只有 c l-珠蛋白基因， 故只有低熔 点的峰。 因此， 自淬灭探针溶解曲栽法可以用于基因分型， 只要 根据熔点峰的有无及熔点的高低就可以对不同的基因型进行区 分。
比较线性自淬灭探针和发夹型自淬灭^针的 PCR-熔解曲线分 析结果，可以看出，发夹型自淬灭探针在低温区的背景信号低， 而 线性自淬灭探针在低温区有明显的噪音峰， 该结果说明了发夹型 自淬灭探针的优势。 实施例 9. 相同荧光标记的两种探针同时检测两种突变的基因型 对于同时存在的但距离较远的突变需要使用不同的探针进行 检测， 通过使用相同荧光标记的不同探针， 但是彼此的熔点有所 差别， 也可在同一个反应管中也能实现对不同区域突变的检测。 以检测 β-珠蛋白的两种突变 -28 (A>G)突变和 IVS- 2-654 (OT)突 变的检测为例加以说明。
我们针对两种突变的野生型分别设计两条探针， 两者 Tm值差 异较大，故两探针与野生型靶杂交时， 具有距离熔点峰相距较远， 很容易分辨。 若模板中含有突变， 则会出现新的熔点峰， 依靠新 出现的熔点峰的 Tm值大小， 可以对不同区域的突变进行区分。 自 淬灭发夹探针 P1与野生型靶完全匹配，故与 -28 (A>G)突变型靶杂 交的 Tm值较低； 自淬灭发夹探针 P8与 IVS- 2-654 (OT)突变型的 靶完全匹配，故与野生型的靶杂交的 Tm值较低。 PCR扩增体系为： 25yL反应体系中含有 lxSSP buffer [67 mM Tris-HCl, H 8.0, 16.6 mM (NH4) 2S04， 6.7mMEDTA, 0.085 mg/mL BSA] , 2.0mMMgCl2, 0.2 mM dNTPs, 1 U Taq (HS) (TAKARA公司， 为热启动 Taq酶， 具有 5'→3'外切酶活性） ， O. l MFl, 0·8μΜ引物 Rl， 0.05 μ Μ 引物 F3， 0.4μΜ 引物 R3， 0.2 μ M 探针 Pl， 0.15μΜ 探针 Ρ8 (各引物和探针的序列见表 2) ， 5 的质粒模板或人类基因组 DNA模板（约为 50 ng) 。 PCR扩增程序程序为： 95 5 min 预 变性， 循环周期为 95 15 s, 52Ό 20 s， 72 20 s， 共 50 个循环，在每个循环退火阶段釆集相应检测通道的荧光信号。 PCR 反应结束后，进行熔解曲线分析，熔解曲线分析程序为： 95°C 变 性 1 min, 35 °C 保温 2 min, 随后按 ΙΓ/step 的升温速率从 40*C 递增至 80Ό 进行熔解曲线分析， 并且釆集相应检测通道 的荧光信号。 实时 PCR 和熔解曲线分析都在 Rotor-Gene 6000 实时 PCR 仪上进行。
结果如图 9 所示， 当模板为野生型（W)时， 探针 P1 和 P8 都 只有与野生型靶杂交的熔点，其中 P1与野生型靶杂交形成双链杂 交体的 Tm值为 67.92"C， P8 与野生型靶杂交形成双链杂交体的 T m 值为 53.27 Π ; 当模板为 IVS-2-654 (C>T)纯合突变型 ( IVS-2-654 M)时， P8探针的 Tm值产生了变化， 为 56.6 , 而 P1 探针的靶仍为野生型， Tm值保持不变； 当模板为- 28(A>G)纯 合突变型（-28M)时， P1探针的 Tm值产生了变化， 为 60.97"C， 而 P8 探针的靶仍为野生型， Tm值保持不变。 因此， 根据各探针 熔点的变化， 我们可以识别不同区域的突变， 从而实现了同种荧 光标记的不同探针对不同区域突变的检测。 实施例 10. 不同荧光标记的发夹型自淬灭探针同时检测多个突 变的基因型
利用不同荧光标记的自淬灭探针， 可以单管检测多个突变， 本 实施例描述了在同一反应管内， 用五种不同荧光标记的发夹型自 淬灭探针检测 β-珠蛋白的多个突变， 即： FAM标记的探针 P1检测 -28(A>G), -29(A>G)两种突变, R0X标记的 P2检测 CD17 (A>T)， CD15/16(+G)和 CD14八 5(+G)三种突变， CAL Fluor Red 635标记 的 P3检测 IVS-1-1 (G>T)， IVS-1-5 (G>C)以及 CD26 (G>A)三个突 变， HEX标记的 P4检测 CD41/42(- TCTT)突变和 CD43(G>T)突变， Quasar 705标记的 P5检测 CD71/72 ( +A ) 和 CD71/72 (+T)两种突 变。 将这五种探针和两对引物即 Fl， Rl, F2 ， R2混合（引物和 探针序列见表 2) ， 可以实现对所有突变基因型的单管同时检测。
PCR 扩增体系为： 25 反应液中含有 l xSSP buffer, 3.0 mM MgCl2， 0.2 mM dNTPs, 1 U Taq (HS) , 0.1 μ M Fl, 1. ΟμΜ Rl, 0.2μΜ F2, 1.6μΜ R2, 0.2 μΜ Pl， 0·2μΜ Ρ2， 0.1 μΜ Ρ3, 0.3μΜΡ4, 0· 1μΜΡ5，5 μ L的人类基因组 DNA模板（约 50 ng )。 PCR的扩增程序和熔解曲线分析程序同实施例 9。
图 10给出了代表性的检测结果， 每一个探针所覆盖的突变的 基因型都得到了正确的检测。 表 2.实施例 9和实施 10所用引物和探针列表
Figure imgf000035_0001
*下划线表示与靶序列互补的序列，小写字母为添加的靶序列无关 序列。 参考文献：
1. 美国专利， US 2006/0019253 A1.
2. 美国专利， US 2003/0224434 A1.
3. 美国专利， US 7, 160, 998 B2. 美国专利， US 6, 472, 156 Bl.
美国专利， US 6, 140, 054.
美国专利， US 6, 635, 427 B2.
美国专利， US 2008/0311579 Al.
美国专利， US 2007/0020665 Al.
美国专利， US 7, 179, 589 B2.
Wittwer C. T. , et al, BioTechniques, 1997, 22: 130- -138.
Ririe K. M. , et al, Anal. Biochem, 1997, 245: 154- -160.
Wittwer CT， et al, Clin Chem, 2003， 49 (6): 853- -860.
Afonina, I. A. , et al, Biotechniques, 2002， 32：
944, 946-949.
Lukhtanov, E. A.， et al, Nucleic Acids Res, 2007

Claims

权 利 要 求
1. (优选用于溶解曲线分析的） 用于检测靶核酸序列变异 的（自淬灭）核酸探针， 该探针标记了荧光基团和淬灭基团， 使得 与不存在靶核酸序列的情况相比， 该探针和靶核酸序列杂交时荧 光 （或荧光强度）增加； 且
该探针可以 （但不必须） 带有能够抵抗 DNA聚合酶的核酸外 切酶活性的修饰，
优选地， 其中：
在所述探针的 5'末端标记荧光基团而在 3'末端标记淬灭基 团，或在所述探针的 3'末端标记荧光基团而在 5'末端标记淬灭基 团；
优选的是， 所述探针的序列包含或者是野生型或变异靶核酸 序列的完全互补序列， 或与所述野生型或变异靶核酸序列的完全 互补序列相比有若干 （例如 1 - 10个， 1-5个, 1-4个， 1-3个， 1-2 个， 1 个或 2 个）错配， 例如具有一个或多个 （例如 1 - 10 个， 1-5个， 1-4个， 1-3个， 1-2个， 1个或 2个）单碱基的转 换、 颠换、 插入和 /或缺失的序列。
2. 权利要求 1 的探针， 其中所述荧光基团包括或者选自下 列标记物： ALEX-350, FAM、 VIC, TET、 CAL Fluor® Go ld 540、 JOE, HEX, CAL F luor® Orange 560、 TAMRA、 CAL F luor® Red 590、 R0X、 CAL Fluor® Red 610、 TEXAS RED, CAL F luor® Red 635、 Qua sar 670、 CY3、 CY5、 CY5. 5和 /或 Quasar 705。
3. 权利要求 1 的探针， 其中所述淬灭基团包括或者选自下 列淬灭剂： DABCYL、 BHQ类淬灭剂 (如 BHQ-1或 BHQ-2 ) 、 ECLIPSE 和 /或 TAMRA。
4. 权利要求 1 的探针， 其中所述探针由未经修饰的碱基组 成。
5. 权利要求 1 的探针， 其中所述探针包含能增强或减弱探 针结合能力的碱基。
6. 权利要求 5 的探针， 其中所述能增强探针结合能力的碱 基包括锁定核酸碱基。
7. 权利要求 5 的探针， 其中所述能减弱探针结合能力的碱 基包括通用结合碱基 I。
8. 权利要求 1 的探针， 其中所述探针带有能够抵抗 DNA聚 合酶的 5'→ 3'核酸外切酶活性的修饰， 优选地， 所述探针的 5' 端能够抗 DM聚合酶的 5'→ 3 '核酸外切酶活性， 所述修饰可以是 例如： 修饰 5'端碱基（例如数个碱基之间， 例如第 1和第 2个碱 基之间）之间的连接、 釆用经修饰的碱基衍生物或添加化学官能 团。
9. 权利要求 8的探针， 其中所述修饰 5'端碱基之间的连接 包括采用硫代磷酸化连接、 甲基磷酸键连接、 硼酸磷酸化连接和 肽核酸连接。
10. 权利要求 9的探针， 其中所述修饰 5'端碱基之间的连接 是在位于 5'端的第一个碱基和第二个碱基之间釆用硫代磷酸化 连接。
11. 权利要求 8 的探针， 其中所述釆用修饰的碱基衍生物包 括采用锁定核酸。
12. 权利要求 1 的探针， 其中所述探针带有能够抵抗 DNA聚 合酶的 3' 5'核酸外切酶活性的修饰， 优选地， 所述探针的 3' 端能够抗 DNA聚合酶的 3' 核酸外切酶活性， 所述修饰可以是 例如： 修饰 3'端碱基（例如数个碱基之间， 例如第 1和第 2个碱 基）之间的连接、 釆用经修饰的碱基衍生物或添加化学官能团。
13. 权利要求 12的探针， 其中所述修饰 3'端碱基之间的连接 包括采用硫代磷酸化连接、 甲基磷酸键连接、 硼酸礫酸化连接和 肽核酸连接。
14. 权利要求 13的探针， 其中所述修饰 3'端碱基之间的连接 是在位于 3， 端的数个碱基之间 （如第一个碱基和第二个碱基之 间） 釆用疏代磷酸化连接。
15. 权利要求 12的探针， 其中所述采用修饰的碱基衍生物包 括采用锁定核酸。
16， 权利要求 1-15 中任一项的探针， 其中所述探针是线性 探针， 其熔点不低于扩增所用引物的熔点， 其中所述探针的长度 为 5- 100个碱基， 例如 10 -100个， 10-50个， 15到 50个， 20 到 50个， 10- 40个碱基， 再例如 10 - 20, 20 - 30， 30 - 40, 15 - 30, 20 - 40个， 15-25个。
17. 权利要求 1-15 中任一项的探针， 其中所述探针是发夹 结构探针， 既可以是天然发夹结构探针也可以是人工发夹结构， 但优选是人工发夹结构探针， 即通过在探针末端人为添加靶序列 无关碱基形成人工发夹结构。添加这种靶序列无关碱基的规则是， 所形成的发夹结构中的臂序列中有部分或者全部碱基与靶序列互 补， 并且形成的臂长一般优选在 2-15个碱基，优选 3-7个碱基之 间， 更优选的是 4 - 7个或 4-6个碱基之间； 其中该探针与靶序列 杂交熔点不低于扩增所用引物的熔点， 其中所述探针的长度为 5 -100个碱基， 例如 10- 100个， 10-50个， 15到 50个， 20到 50个， 10-40个碱基， 再例如 10-20， 20 - 30, 30 - 40, 15 - 30, 20 - 40个， 15-25个。
18. 试剂盒，其含一个或多个权利要求 1- 16中任一项的探针， 其中当有多个探针时，各探针上的荧光基团相同或不同，任选地， 所述试剂盒还包含扩增引物和 /或核酸聚合酶和 /或扩增反应所需 其它成分， 所述试剂盒可用于对待测靶基因进行核酸扩增并用熔 解曲线分析进行靶序列变异分析。
19. 用于检测核酸是否存在变异例如单核苷酸变异或变异类 型的方法， 包括：
( 1 )针对需要检测核酸序列变异的核酸制备权利要求 1 - 17 中任一项的探针；
( 2 )扩增（例如通过 PCR)含有待检测核酸的片段， 在扩增前、 扩增中或者扩增之后 （优选在扩增前） 在扩增反应中加入所述探 针；
( 3 )扩增结束后对步骤（ 2 )所获得的含有所述探针的扩增产 物进行熔解曲线分析， 根据自淬灭探针所对应的熔点 （或者所述 探针和待检测核酸序列之间杂交体的熔点） 或者该熔点的差异来 判断待检测核酸是否具有变异以及任选地判断可能的变异类型； 优选地， 步骤（2 ) 所获得的扩增产物中包含足量的完整探针 用于熔解曲线分析；
优选地， 步骤（2 )所说的扩增是不对称 PCR, 即反应混合物 中一种 PCR扩增引物相对过量，它所延伸而产生的链与探针杂交； 优选的， 所述扩增反应中包含参照核酸或者野生型核酸。
20. 权利要求 19的方法， 其中， 所述探针带有能够抵抗 DNA 聚合酶的核酸外切酶活性的修饰， 和 /或， 所述探针不带有能够抵 抗 DNA聚合酶的核酸外切酶活性的修饰， 但所述扩增用酶不具有 核酸外切酶活性或仅有足够低的核酸酶外切活性， 使得步骤（2 ) 所获得的扩增产物中包含足量的完整探针用于熔解曲线分析。
21. 权利要求 19 的方法， 其中， 步骤（2 ) 中， 在扩增前加 入所述探针， 所述探针带有或者不带有能够抵抗 DNA聚合酶的核 酸外切酶活性的修饰， 所说的扩增使用没有外切活性或者外切活 性很低的耐热核酸聚合酶， 以保证预先加入的探针在 PCR反应后 仍有足量的完整探针用于熔解曲线分析。
22. 用于检测核酸是否存在变异例如单核苷酸变异或变异类 型的方法， 包括：
( 1 )针对基因组中一个或多个待检是否各包含一种或多种具 有靶序列变异例如单核苷酸变异的待检等位核酸序列的核酸区 段， 制备能够覆盖所述待检核酸区段的数量的权利要求 1 - 17中 任一项的探针或含所述各探针的一个或多个权利要求 18 的试剂 盒， 并于 PCR反应开始前将所述探针或所述试剂盒加入下述（2 ) 的反应体系中；
( 2 )在 PCR反应体系中扩增基因组中所述一个或多个待检是 否各包含一种或多种具有所述靶序列变异例如单核苷酸变异的所 述待检等位核酸序列的核酸区段；
( 3 )仍于所述反应体系中，在逐步升温下或者在逐步降温下 监控步骤（1 ) 中制备的各探针与步骤（2 ) 中扩增的各待检等位 核酸序列的相互作用所致的荧光变化， 从而同时得到所述各探针 所对应的熔解曲线；
( 4 )对通过步骤（ 3 )得到的熔解曲线求导， 并取其负导数 ( -dF/dT ) ， 从而得到所述各探针所对应的熔点； 及
( 5 )通过比较步骤（4 )得到的对应于各待检等位核酸序列 和各探针之间的熔解温度来分析各待检等位核酸序列间是否存在 靶序列变异例如单核苷酸变异，
其中所述单核苷酸变异可以是同一物种的不同个体间的同一 基因座位上的核酸序列中的一个或多个相同或不同位置的单碱基 的转换、 颠换、 插入或缺失， 且所述基因组的一个或多个核酸区 段相同或不同。
优选地， 步骤 （2 ) 所获得的扩增产物中包含足量的完整探针 用于熔解曲线分析；
优选地， 步骤（2 )所说的扩增是不对称 PCR, 即反应混合物 中一种 PCR扩增引物相对过量，它所延伸而产生的链与探针杂交； 优选的， 所述扩增反应中包含参照核酸或者野生型核酸。
23. 权利要求 22的方法， 其中， 所述探针带有能够抵抗 DNA 聚合酶的核酸外切酶活性的修饰， 和 /或， 所述探针不带有能够抵 抗 DNA聚合酶的核酸外切酶活性的修饰， 但所述扩增用酶不具有 核酸外切酶活性或仅有足够低的核酸酶外切活性， 使得步骤（2 ) 所获得的扩增产物中包含足量的完整探针用于熔解曲线分析。
24. 权利要求 1 - 17中任一项的探针或权利要求 18的试剂盒 用于检测核酸变异例如是否存在单核苷酸变异或变异类型的用 途， 其中例如所述单核苷酸变异可以是同一物种的不同个体间的 同一基因座位上的核酸序列中的一个或多个相同或不同位置的单 碱基的转换、 颠换、 插入或缺失。
25. 一种利用自淬灭探针通过核酸扩增溶解曲线分析检测靶 序列变异或变异类型的方法， 该方法使用的自淬灭探针的序列包 含或者是： 野生型或变异靶核酸序列的完全互补序列， 或与所述 野生型或变异靶核酸序列的完全互补序列相比有若干 （例如 1到 10个， 1到 5个， 1到 3个， 1到 2个， 1个或 2个）错配， 例如 具有一个或多个单碱基（例如 1到 10个， 1到 5个， 1到 3个， 1 到 2个， 1个或 2个） 的转换、 颠换、 插入和 /或缺失，
所述自淬灭探针标记了荧光基团和淬灭基团， 使得与不存在 靶核酸序列的情况相比， 该探针和靶核酸序列杂交时荧光（或荧 光强度）增加；
该方法包括： 在扩增反应液中预先加入自淬灭探针， 在适当 的反应条件下扩增， 之后进行熔解曲线分析， 根据自淬灭探针与 靶核酸杂交体的熔点来分析是否存在变异和任选地变异的类型， 所述的反应条件是保证在核酸扩增后， 有足量的完整的探针 和足量的单链耙序列之间可以进行熔解曲线分析的条件，
优选地， 所述探针是权利要求 1 - 17任一项的探针。
PCT/CN2010/000753 2009-05-26 2010-05-26 一种检测核酸序列变异的方法 WO2010135917A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP10779999.1A EP2436777B1 (en) 2009-05-26 2010-05-26 Method for detecting variations in nucleic acid sequences
US13/322,716 US8691504B2 (en) 2009-05-26 2010-05-26 Method for detecting variations in nucleic acid sequences
CN201080023112.9A CN102449167B (zh) 2009-05-26 2010-05-26 一种检测核酸序列变异的方法
US14/182,067 US9334533B2 (en) 2009-05-26 2014-02-17 Method for detecting variations in nucleic acid sequences

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200910143480.6 2009-05-26
CN200910143480 2009-05-26

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US13/322,716 A-371-Of-International US8691504B2 (en) 2009-05-26 2010-05-26 Method for detecting variations in nucleic acid sequences
US14/182,067 Continuation US9334533B2 (en) 2009-05-26 2014-02-17 Method for detecting variations in nucleic acid sequences
US14/182,067 Division US9334533B2 (en) 2009-05-26 2014-02-17 Method for detecting variations in nucleic acid sequences

Publications (1)

Publication Number Publication Date
WO2010135917A1 true WO2010135917A1 (zh) 2010-12-02

Family

ID=43222138

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2010/000753 WO2010135917A1 (zh) 2009-05-26 2010-05-26 一种检测核酸序列变异的方法

Country Status (4)

Country Link
US (2) US8691504B2 (zh)
EP (1) EP2436777B1 (zh)
CN (1) CN102449167B (zh)
WO (1) WO2010135917A1 (zh)

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102449167B (zh) * 2009-05-26 2014-12-17 厦门大学 一种检测核酸序列变异的方法
CN103228785B (zh) * 2010-11-24 2016-09-07 株式会社钟化 扩增核酸的检测方法和检测装置
EP2766711B1 (en) * 2011-10-14 2017-12-13 Becton, Dickinson and Company Square wave thermal cycling
CN104271767B (zh) 2012-02-03 2017-08-25 加州理工学院 多路生化测定中信号的编码和解码
US9791372B2 (en) 2012-08-03 2017-10-17 California Institute Of Technology Multiplexing and quantification in PCR with reduced hardware and requirements
EP2708895B1 (en) 2012-08-21 2019-05-08 Industrial Technology Research Institute Method for identifying more than one target nucleic acid contained in a sample
CN104404159A (zh) * 2014-12-10 2015-03-11 苏州旷远生物分子技术有限公司 用于检测人hla-b*5801基因的引物、探针、荧光pcr试剂盒和方法
KR102266987B1 (ko) * 2014-12-24 2021-06-21 주식회사 시선바이오머티리얼스 Pna 프로브를 이용한 인공합성 올리고의 품질보증 방법
CA3012815C (en) * 2016-02-09 2021-09-28 Eiken Kagaku Kabushiki Kaisha Method for detecting target nucleic acid and nucleic acid probe used therein
EP3472354A4 (en) 2016-06-17 2020-01-01 California Institute of Technology NUCLEIC ACID ACTIONS AND RELATED PROCEDURES AND COMPOSITIONS
CA3042691A1 (en) * 2016-11-03 2018-05-11 Youhealth Biotech, Limited Methods and compositions for cellular reprogramming
CN107099583A (zh) * 2017-04-05 2017-08-29 浙江大学 一种基于分子信标溶解曲线的可用于核酸扩增的快速、特异性检测方法
CN107034277B (zh) * 2017-04-27 2021-02-05 厦门大学 一种检测低丰度基因突变的方法
CN107488715A (zh) * 2017-08-14 2017-12-19 福建医科大学孟超肝胆医院 基于自淬灭探针熔解曲线的pnpla3易感基因检测试剂盒及方法
CN107267657A (zh) * 2017-08-15 2017-10-20 江西贤聚景欣医药生物科技有限公司 检测靶核酸序列的引物、荧光探针及方法
CN111321211A (zh) * 2018-12-14 2020-06-23 北京大学深圳医院 人β地中海贫血基因分型检测的试剂及检测方法
CN112301120A (zh) * 2019-07-29 2021-02-02 上海利康精准医疗技术有限公司 一种用于检测adrb1基因多态性的探针、引物及试剂盒
CN114686573B (zh) * 2020-12-25 2024-04-12 厦门大学 一种检测靶核酸拷贝数重复的方法和试剂盒
CN112921120B (zh) * 2021-03-01 2022-07-05 北京化工大学 一种可重复利用的核酸荧光探针及相应的核酸检测方法
CN114317699B (zh) * 2021-12-23 2023-01-10 郑州华之源医学检验实验室有限公司 一种基于熔解曲线正负峰形分析的多重pcr检测方法及应用
WO2024000312A1 (zh) * 2022-06-29 2024-01-04 深圳华大生命科学研究院 碱基识别方法及系统、基因测序仪及存储介质
CN115948609B (zh) * 2022-08-05 2023-10-20 深圳市儿童医院 检测SARS-CoV-2的组合物及其试剂盒和应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6140054A (en) 1998-09-30 2000-10-31 University Of Utah Research Foundation Multiplex genotyping using fluorescent hybridization probes
US6472156B1 (en) 1999-08-30 2002-10-29 The University Of Utah Homogeneous multiplex hybridization analysis by color and Tm
US6635427B2 (en) 2000-08-11 2003-10-21 University Of Utah Research Foundation Single-labeled oligonucleotide probes for homogeneous nucleic acid sequence analysis
US20030224434A1 (en) 2002-05-13 2003-12-04 Wittwer Carl T. Genotyping by amplicon melting curve analysis
US20060019253A1 (en) 2002-10-23 2006-01-26 Wittwer Carl T Amplicon melting analysis with saturation dyes
US7160998B2 (en) 1996-06-04 2007-01-09 University Of Utah Research Foundation Monitoring amplification with fret probes
CN1900312A (zh) * 2006-07-24 2007-01-24 厦门大学 实时pcr检测中抗高温聚合酶核酸外切酶活性的荧光探针
US20070020665A1 (en) 2005-06-30 2007-01-25 Roche Molecular Systems Probes and methods for hepatitis C virus typing using single probe analysis
US7179589B2 (en) 1998-10-08 2007-02-20 Dynametrix Ltd. Detection of nucleic acid polymorphism
US20080311579A1 (en) 2000-03-29 2008-12-18 David John French Hybridisation beacon and method of rapid sequence detection and discrimination

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0826066B1 (en) * 1995-05-05 2000-09-13 The Perkin-Elmer Corporation Methods and reagents for combined pcr amplification and hybridization probing assay
US20050112637A1 (en) * 1995-09-08 2005-05-26 Chatterjee Deb K. Mutant DNA polymerases and uses thereof
AUPP844899A0 (en) 1999-02-01 1999-02-25 University Of Western Australia, The A method for detecting methylated nucleic acids
US6958390B1 (en) * 2000-06-14 2005-10-25 Stratagene California Compositions and kits for herpes simplex virus type 1 and 2 nucleic acid detection
US6852491B2 (en) * 2001-09-04 2005-02-08 Abbott Laboratories Amplification and detection reagents for HIV-1
CA2472554A1 (en) * 2002-02-27 2003-09-04 Biosource International Inc. Methods of using fet labeled oligonucleotides that include a 3' 5' exonuclease resistant quencher domain and compositions for practicing the same
EP1426448A1 (en) * 2002-12-03 2004-06-09 bioMérieux BV Method for lowering the effects of sequence variations in a diagnostic hybridization assay, probe for use in the assay and assay
US7718361B2 (en) * 2002-12-06 2010-05-18 Roche Molecular Systems, Inc. Quantitative test for bacterial pathogens
CA2494571C (en) * 2003-12-02 2010-02-09 F.Hoffmann-La Roche Ag Oligonucleotides containing molecular rods
US7476733B2 (en) * 2005-03-25 2009-01-13 The United States Of America As Represented By The Department Of Health And Human Services Development of a real-time PCR assay for detection of pneumococcal DNA and diagnosis of pneumococccal disease
US7553955B2 (en) * 2005-09-01 2009-06-30 The Trustees Of The University Of Pennsylvania Molecular beacons, methods and kits for detecting DNA damage response
WO2007058898A2 (en) * 2005-11-10 2007-05-24 Panomics, Inc. Detection of nucleic acids through amplification of surrogate nucleic acids
JP4713514B2 (ja) * 2006-02-20 2011-06-29 エフ．ホフマン−ラ ロシュ アーゲー 改善された標識試薬
EP1989324B1 (en) * 2006-02-27 2011-05-18 Roche Diagnostics GmbH Pcr hot start by magnesium sequestration
WO2007115207A2 (en) * 2006-03-31 2007-10-11 Regents Of The University Of Minnesota Irf-5 haplotypes in systemic lupus erythematosus
DK2134861T3 (da) 2007-03-08 2012-07-30 Idaho Technology Fremgangsmåde for smelteanalyse af nukleinsyrer
US20100240049A1 (en) * 2009-01-16 2010-09-23 Cepheid Methods of Detecting Cervical Cancer
US20100233704A1 (en) * 2009-02-25 2010-09-16 Cepheid Methods of detecting lung cancer
CN102449166B (zh) 2009-05-26 2013-12-04 厦门大学 一种单管检测多个单核苷酸变异或单核苷酸多态性的方法
CN102449167B (zh) * 2009-05-26 2014-12-17 厦门大学 一种检测核酸序列变异的方法
US20120237451A1 (en) * 2009-09-18 2012-09-20 Antony Kuang-Shih Chen Novel molecular beacons
WO2011069160A1 (en) * 2009-12-05 2011-06-09 Evogen, Inc. Methods of quantifying nucleic acids
AU2012308365A1 (en) * 2011-09-16 2014-04-24 Cepheid Methods of detecting sepsis

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7160998B2 (en) 1996-06-04 2007-01-09 University Of Utah Research Foundation Monitoring amplification with fret probes
US6140054A (en) 1998-09-30 2000-10-31 University Of Utah Research Foundation Multiplex genotyping using fluorescent hybridization probes
US7179589B2 (en) 1998-10-08 2007-02-20 Dynametrix Ltd. Detection of nucleic acid polymorphism
US6472156B1 (en) 1999-08-30 2002-10-29 The University Of Utah Homogeneous multiplex hybridization analysis by color and Tm
US20080311579A1 (en) 2000-03-29 2008-12-18 David John French Hybridisation beacon and method of rapid sequence detection and discrimination
US6635427B2 (en) 2000-08-11 2003-10-21 University Of Utah Research Foundation Single-labeled oligonucleotide probes for homogeneous nucleic acid sequence analysis
US20030224434A1 (en) 2002-05-13 2003-12-04 Wittwer Carl T. Genotyping by amplicon melting curve analysis
US20060019253A1 (en) 2002-10-23 2006-01-26 Wittwer Carl T Amplicon melting analysis with saturation dyes
US20070020665A1 (en) 2005-06-30 2007-01-25 Roche Molecular Systems Probes and methods for hepatitis C virus typing using single probe analysis
CN1900312A (zh) * 2006-07-24 2007-01-24 厦门大学 实时pcr检测中抗高温聚合酶核酸外切酶活性的荧光探针

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
AFONINA, I.A. ET AL., BIOTECHNIQUES, vol. 32, 2002, pages 940 - 944,946-949
AFONINA, LA. ET AL., BIOTECHNIQUES, vol. 32, 2002, pages 940 - 944,946-949
HUANG QIUYING: "Study on Real-Time PCR-Based Approaches for Multiple Targets Detection", CHINESE MASTER'S THESES FULL-TEXT DATABASE, BASIC SCIENCES, vol. 12, 15 December 2009 (2009-12-15), pages 1 - 132, XP008165773 *
JESSE MONTGOMERY ET AL.: "Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis", NATURE PROTOCOLS, vol. 2, no. 1, 22 February 2007 (2007-02-22), pages 59 - 66, XP008165524 *
LUKHTANOV, E.A. ET AL., NUCLEIC ACIDS RES, vol. 35, 2007, pages E30
LUMING ZHOU ET AL.: "High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution", MOLECULAR DIAGNOSTICS AND GENETICS, vol. 51, no. 10, 2005, pages 1770 - 1777, XP008118572 *
RIRIE K, M. ET AL., ANAL. BIOCHEM, vol. 245, 1997, pages 154 - 160
RIRIE K. M. ET AL., ANAL. BIOCHEM, vol. 245, 1997, pages 154 - 160
See also references of EP2436777A4
WITTWER C. T. ET AL., BIOTECHNIQUES, vol. 22, 1997, pages 130 - 138
WITTWER C. T. ET AL., CLIN CHEM, vol. 49, 2003, pages 853 - 860
WITTWER CT ET AL., CLIN CHEM, vol. 49, no. 6, 2003, pages 853 - 860

Also Published As

Publication number Publication date
EP2436777A1 (en) 2012-04-04
US20120077195A1 (en) 2012-03-29
CN102449167B (zh) 2014-12-17
EP2436777A4 (en) 2012-11-07
CN102449167A (zh) 2012-05-09
US9334533B2 (en) 2016-05-10
US8691504B2 (en) 2014-04-08
EP2436777B1 (en) 2015-08-19
US20140162898A1 (en) 2014-06-12

Similar Documents

Publication Publication Date Title
WO2010135917A1 (zh) 一种检测核酸序列变异的方法
AU2007307320B2 (en) Nucleic acid size detection method
EP1423532B1 (en) Nucleic acid amplification method
US6270967B1 (en) Method for detecting a nucleic acid base sequence
US8916349B2 (en) Assay method for target nucleic acid by signal amplification using probe hybridization and restriction
JP5424903B2 (ja) 融解解析のためのプライマー
JP2003528626A (ja) ハイブリダイゼーション・ビーコン、並びに迅速に配列を検出および判別する方法
EP1633888A2 (en) Means and method for the detection of target nucleotide sequences using ligation assays with improved oligonucleotide probe pairs
WO2010135916A1 (zh) 一种单管检测多个单核苷酸变异或单核苷酸多态性的方法
KR102488559B1 (ko) 가닥-침투 기반 dna 증폭방법
GB2312747A (en) Primers with non-complementary tails for detection of diagnostic base sequences
EP3055430A2 (en) Multiplex probes
US20140377762A1 (en) Method for enriching and detection of variant target nucleic acids
CN111100908A (zh) 一种检测核苷酸片段缺失的方法和试剂盒
US20050053957A1 (en) Polynucleotide sequence detection assays
CN105705660B (zh) 使用重叠引物和熔解探针检测单核苷酸多态性
WO2022126721A1 (zh) 一种高特异性的检测靶核酸序列的方法
WO2021128860A1 (zh) 一种不对称扩增靶核酸的方法
WO2021128823A1 (zh) 一种不对称扩增多个靶核酸的方法
CN114686574B (zh) 一种检测核苷酸大片段缺失的方法和试剂盒

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201080023112.9

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10779999

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 13322716

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 13322716

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2010779999

Country of ref document: EP