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
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • 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

Definitions

• the present invention relates to a method for detecting nucleotide variations using labeled primers.
• HBV hepatitis B virus
• direct sequencing after PCR electrophoresis after restriction enzyme digestion
• PCR-RFLP Restriction Fragment Mass Polymorphism
• LightCycler probe hybridization and primer-specific real-time PCR.
• the conventional methods have serious problems in terms of accuracy or convenience.
• nucleotide variation could not be detected with a desired accuracy.
• at least two nucleotide variations could not reliably detected in a simultaneous manner.
• the present inventors have developed primers having unique structures and found that at least two nucleotide variations could be detected without false results in more convenient manner, eventually accomplishing the present invention.
• a method for simultaneously detecting at least two nucleotide variations of a target nucleotide sequence in a nucleic acid sample containing the target nucleotide sequence with the nucleotide variation which comprises the steps of:
• a target specific primer 1 hybridizable with a region of the target nucleotide sequence positioned at upstream of the variation- occurring region to be hybridized with the NVS-Pl primer
• a second nucleotide variation specific primer 2 hybridizable with a second variation-occurring region of the target nucleotide sequence having a nucleotide corresponding to a second nucleotide variation that is positioned at a site same as, adjacent to or distant from the first nucleotide variation, wherein the NVS-P2 primer comprises a nucleotide corresponding to the second nucleotide variation and links with a detectable label at 5' end;
• a target specific primer 2 (TSP2) hybridizable with a region of the target nucleotide sequence positioned at downstream of the second variation-occurring region to be hybridized with the NVS-P2 primer; (b) performing at least two cycles of primer annealing, primer extending and denaturing using the primers to amplify the target sequence; (c) hybridizing the amplified product of the step (b) with a first probe and a second probe, wherein the first probe hybridizes specifically with an amplified product by the primers (i) and (ii) in the step (b) and the second probe hybridizes specifically with an amplified product by the primers (iii) and (iv) in the step (b); and (d) detecting a hybridization signal from the step (c).
• TSP2 target specific primer 2
• the present invention is drawn to provide methods for simultaneously detecting various nucleotide variations or SNP (single nucleotide polymorphism) in target sequences.
• the present invention provides methods for simultaneously detecting nucleotide variations at the same (single) nucleotide or in the same (single) codon.
• the present invention is a very profitable method which can automate a whole process.
• nucleotide variation refers to various alleles at same
• nucleotide variation includes mutant type as well as wild type. Therefore, the application object of the present invention is "a detection method for nucleotide variation", presented by “genotyping” or "a detection method for allele”.
• the nucleotide sequences to be detected by the present invention include gDNA, cDNA and RNA.
• the NVS primers used in this invention have the structure represented by the following general formula I:
• X represents a detectable label
• Ap represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the target nucleotide sequence
• Yq represents a separation portion comprising at least three universal bases
• Vr represents a variation specificity portion having a nucleotide complementary or corresponding to the nucleotide variation and a nucleotide sequence substantially complementary to the target nucleotide sequence
• p, q and r represent the number of nucleotides
• A, Y, and V are deoxyribonucleotide or ribonucleotide
• Tm of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest Tm in the three portions
• the separation portion separates the variation adjacent specificity portion from the variation specificity portion in terms of hybridization specificity, whereby the hybridization specificity of the primers are determined dually by the variation adjacent specificity portion and the variation specific
• oligonucleotides having such structure are named as dual specificity oligonucleotides (DSO).
• DSO dual specificity oligonucleotides
• the NVS primers used in this invention are modification of the DSO and have a unique structure represented by the general formula I.
• the NVS primers comprise a variation adjacent specificity portion, a separation portion and a variation specificity portion.
• the variation adjacent specificity portion and the variation specificity portion are physically and functionally separated by the separation portion such that the overall hybridization specificity of primers is dually determined by the variation adjacent specificity portion and the variation specificity portion, finally resulting in dramatically enhancing the overall hybridization specificity of primers.
• NVS primers when NVS primers are annealed to variation-containing target sequences, their specificity is dually determined by both the variation adjacent specificity portion and the variation specificity portion rather than determined by the overall length of primers.
• the NVS primers are annealed to target sequences in a different performance from conventional primers, which results in greatly increasing the annealing specificity.
• the universal base in the separation portion is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2'- deoxyinosine, 2-aza-2'-deoxyinosine, 2'-0Me inosine, 2'-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2'-0Me 3-nitropyrrole, 2'-F 3-nitropyrrole, l-(2'-deoxy-beta-D- ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole, 5-nitroindole, 2'-0Me 5-nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4- aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2'-F nebularine, 2'-F 4- nitrobenzimidazole,
• the universal base or non-discriminatory base analog is deoxyinosine, l-(2'-deoxy-beta-D- ribofuranosyl)-3-nitropyrrole or 5-nitroindole, most preferably, deoxyinosine. It is preferable that the separation portion comprises contiguous nucleotides having at least three universal bases.
• the variation adjacent specificity portion is longer than the variation specificity portion.
• the variation adjacent specificity portion is preferably 15-40 nucleotides, more preferably 15-25 nucleotides in length.
• variation specificity portion is 3-15 nucleotides, more preferably 6-13 nucleotides in length.
• the separation portion is preferably 3-10 nucleotides, more preferably 4-8 nucleotides, most preferably 5-7 nucleotides in length.
• the Tm of the variation adjacent specificity portion ranges from 40°C to 80 0 C, more preferably 45 0 C to 65°C.
• the Tm of the variation specificity portion ranges preferably from 10 0 C to 40 0 C. It is preferable that the Tm of the separation portion ranges from 3°C to 15°C.
• the variation specificity portion of primers has a nucleotide complementary or corresponding to the nucleotide variation. Where the primers are annealed to the sense strand of the target nucleotide sequence, the variation specificity portion has a nucleotide complementary to the nucleotide variation. In contrast, where the primers are annealed to the antisense strand of the target nucleotide sequence, the variation specificity portion has a nucleotide corresponding to the nucleotide variation. According to a preferred embodiment, the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at the 3'-end of the variation specificity portion or at 1-10 nucleotides apart from the
• the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at 2-7 nucleotides, more still preferably, 3-6 nucleotides apart from the 3'-end of the variation specificity portion.
• the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at the center or around the center of the variation specificity portion.
• the nucleotide complementary or corresponding to the nucleotide variation is located at 3- 6 nucleotides, preferably 4-5 nucleotides, more preferably 4 nucleotides apart from the 3'-end of the variation specificity portion.
• nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located 3-6 nucleotides apart from the 3'-end of the variation specificity portion or around the center of the variation specificity portion, the following advantages may be obtained:
• the nucleotide variation-specific base is located at the 3'-end of primers. This position is responsible for the slight difference in Tm values of annealing occurrence and non-annealing occurrence (i.e., mismatching) of the nucleotide variation-specific base.
• mismatching i.e., mismatching
• the nucleotide variation-specific base is located around at the center of conventional primers, the difference in Tm values between annealing and non-annealing (i.e., mismatching) becomes larger.
• most of thermostable polymerases used in amplification reactions catalyze the reactions in disregard of mismatching in the central region of primers, resulting in the generation of false- positive results.
• the present invention ensures to completely overcome the shortcoming of conventional techniques described above.
• the Tm value of the variation specificity portion becomes much lower because the mismatching occurs at the center of the variation specificity portion.
• the mismatching event is recognized to occur around the 3'-end of primers and therefore thermostable polymerases do not catalyze reactions. Therefore, mismatching of the nucleotide variation-specific base induces no false-positive results.
• the NVS primers used in this invention have the structure represented by the following general formula II:
• X is a detectable label
• Ap represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the target nucleotide sequence
• (dl)q represents a separation portion comprising contiguous deoxyinosine bases
• Vr represents a variation specificity portion having a nucleotide complementary or corresponding to the nucleotide variation and a nucleotide sequence substantially complementary to the target nucleotide sequence
• p is an integer of 15-25
• q is an integer of 4-8 and r is an integer of 6-13
• the nucleotide complementary or corresponding to the nucleotide variation is located at the center of
• (dl) represents a separation portion comprising contiguous deoxyinosine bases and the number of deoxyinosine residues is 4-8.
• Other bases other than deoxyinosine may be introduced into the separation portion so long as (dl) functions as separation portions.
• the nucleotide complementary or corresponding to the nucleotide variation is located at the center of Vr. For instance, where Vr is 6, 7, 8, 9, 10, 11, 12 and 13 nucleotides in length, the nucleotide complementary or corresponding to the nucleotide variation is located 3-4, 3-5, 4-5, 4-
• primer refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand (template) is induced, i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, and at a suitable temperature and pH.
• the primer is preferably single stranded for maximum efficiency in amplification.
• the primer is an oligodeoxyribonucleotide.
• the primer of this invention can be comprised of naturally occurring dNMP (i.e., dAMP, dGM, dCMP and dTMP), modified nucleotide, or non-natural nucleotide.
• the primer can also include ribonucleotides.
• the primer used in this invention may include nucleotides with backbone modifications such as peptide nucleic acid (PNA) (M.
• PNA peptide nucleic acid
• nucleotides with sugar modifications such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-OaIIyI DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides having base modifications such as C-5 substituted pyrimidines (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-,
• sequences of the primers may comprise some mismatches, so long as they can be hybridized with templates and serve as primers.
• hybridizing refers to the formation of a duplex structure by pairing complementary single stranded nucleic acids.
• the hybridization may occur between single stranded nucleic acids with perfectly matched sequences or between single stranded nucleic acids with some mismatched sequences.
• the sequence complementarity for hybridization depends on hybridization conditions, particularly temperature. Generally, as the temperature for hybridization becomes higher, the perfectly complementary sequences are very likely to be hybridized. In the case that the hybridization temperature becomes lower, some mismatched sequences may be hybridized. As the hybridization temperature becomes lower, the sequence complementarity for hybridization becomes lower.
• Suitable hybridization conditions may be routinely determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength may be varied depending on various factors, including the length and GC content of primer and target nucleotide sequence.
• the detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and M. LM. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.(1999).
• the hybridization (annealing) is performed at temperature of 40-70 0 C, more preferably 45-68°C, still more preferably 50-65 0 C, most preferably 60-65 0 C.
• the present method to detect nucleic acid molecules containing nucleotide variations can detect any variations of interest.
• the nucleic acid molecules include DNA and RNA.
• the nucleic acid molecule may be in either a double-stranded or single- stranded form. Where the nucleic acid as starting material is double-stranded, it is preferred to render the two strands into a single-stranded or partially single-stranded form.
• Methods known to separate strands includes, but not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins. For instance, strand separation can be achieved by heating at temperature ranging from 80 0 C to 105 0 C. General methods for accomplishing this treatment are provided by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).
• a reverse transcription step is necessary prior to performing annealing step, details of which are found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, IM.Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988)).
• an oligonucleotide dT primer hybridizable to poly A tail of mRNA is used.
• the oligonucleotide dT primer is comprised of dTMPs, one or more of which may be replaced with other dNMPs so long as the dT primer can serve as primer.
• Reverse transcription can be done with reverse transcriptase that has RNase H activity. If one uses an enzyme having RNase H activity, it may be possible to omit a separate RNase H digestion step by carefully choosing the reaction conditions.
• the primers used for the present invention is hybridized or annealed to a site on the template such that double-stranded structure is formed.
• Conditions of nucleic acid annealing suitable for forming such double stranded structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).
• DNA polymerases can be used in the extension step of the present methods, which includes "Klenow" fragment of E. coli DNA polymerase I, a thermostable DNA polymerase, and bacteriophage T7 DNA polymerase.
• the polymerase is a thermostable DNA polymerase which may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Many of these polymerases may be isolated from bacterium itself or obtained commercially. Polymerase to be used with the subject invention can also be obtained from cells which express high levels of the cloned genes encoding the polymerase.
• components required for such reaction When a polymerization reaction is being conducted, it is preferable to provide the components required for such reaction in excess in the reaction vessel. Excess in reference to components of the extension reaction refers to an amount of each component such that the ability to achieve the desired extension is not substantially limited by the concentration of that component. It is desirable to provide to the reaction mixture an amount of required cofactors such as Mg2+, dATP, dCTP, dGTP, and dTTP in sufficient quantity to support the degree of the extension desired.
• All of the enzymes used in this amplification reaction may be active under the same reaction conditions. Indeed, buffers exist in which all enzymes are near their optimal reaction conditions. Therefore, the amplification process of the present invention can be done in a single reaction volume without any change of conditions such as addition of reactants.
• Annealing or hybridization in the present method is performed under stringent conditions that allow for specific binding between the primer and the target nucleotide sequence (at this time, the separation portion cannot be annealed to the target nucleotide sequence).
• stringent conditions for annealing will be sequence- dependent and varied depending on environmental parameters.
• the annealing temperature ranges from 40 0 C to 70 0 C, more preferably from 45°C to 68°C, more preferably from 5O 0 C to 65°C, most preferably from 60 0 C to 65 0 C.
• the amplification is performed in accordance with PCR which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.
• the detectable labels linked with NVS primer are, a chemical label ⁇ e.g, biotin), a enzymatic label (alkaline phosphatase, phycoerythrin, peroxidase, ⁇ -galactosidase and ⁇ -glucosidase), a radioactive label
• a fluorescent label HEX, TET, Dabsyl and FAM
• a luminescent label a chemiluminescent label
• a FRET (fluorescence resonance energy transfer) label a heavy metal label (gold).
• the label are a chemical label and a metal label, still more preferably the label are a chemical label, most preferably the label is biotin.
• the enzyme labels are enzymes that catalyze a chromogen reaction, a fluorescence reaction, a luminescence reaction or a infrared reaction.
• Primer labels by a label material are performed, by various methods to be performed ordinarily in the art, for example, nick translation method, random priming method (Multiprime DNA labeling system booket, "Amersham”(1989)) and carnation method (Maxam & Gilbert, Method in Enzymology, 65:499(1986)).
• the target specific primers (TSP) used in this invention are annealed to outer regions of variation-occurring regions to be analyzed, they are designed to have conventional primer structures without the separation portion.
• the target specific primers have a structure represented by the general formula I and the variation specificity portion has a nucleotide sequence substantially complementary to the target nucleotide sequence without the nucleotide complementary or corresponding to the nucleotide variation.
• the step (a) is carried out using at least one of additional nucleotide variation specific primers hybridizable with nucleotide variations other than the nucleotide variations to be hybridized with the NVS-Pl and NVS-P2 primers.
• additional nucleotide variation specific primers hybridizable with nucleotide variations other than the nucleotide variations to be hybridized with the NVS-Pl and NVS-P2 primers.
• additional nucleotide variation specific primers hybridizable with nucleotide variations other than the nucleotide variations to be hybridized with the NVS-Pl and NVS-P2 primers.
• additional nucleotide variation specific primers hybridizable with nucleotide variations other than the nucleotide variations to be hybridized with the NVS-Pl and NVS-P2 primers.
• the amplified product is hybridized with two probes: One of two probes hybridizes only specifically with an amplified product by the primers (i) and (ii) in the step (b) and the other hybridizes only specifically with an amplified product by the primers (iii) and (iv) in the step (b).
• the first and second probes are inevitable ones used in the hybridization reaction, as shown in Fig.l.
• the probes are immobilized on a solid substrate to fabricate microarray. In microarray, the probes serve as hybridizable array elements.
• a preferable substrate includes suitable solid or semi-solid supporters, such as membrane, filter, chip, slide, wafer, fiber, magnetic or nonmagnetic bead, gel, tubing, plate, macromolecule, microparticle and capillary tube.
• the hybridizable array elements are arranged and immobilized on the substrate. Such immobilization occurs through chemical binding or covalent binding such as UV.
• the hybridizable array elements are bound to a glass surface modified to contain epoxi compound or aldehyde group or to a polylysin-coated surface. Further, the hybridizable array elements are bound to a substrate through linkers (e.g. ethylene glycol oligomer and diamine).
• nucleic acid hybridization suitable for the step (c) are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).
• the stringent condition is determined by controlling various factors such as temperature, ionic strength (buffer concentration) and the presence of other compounds such as organic solvents. As understood by those of skill in the art, the stringent conditions are sequence dependent.
• the high stringent condition includes hybridization under 0.5 M NaHPO 4 , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA at 65 0 C, and washing at 68 0 C by use of 0.1 x SSC (standard saline citrate)/0.1% SDS.
• the high stringent condition includes washing at 68 0 C by use of 6 x SSC/0.05% sodium pyrophosphate.
• the low stringent condition includes washing at 42 0 C by use of 0.2 x SSC/0.1% SDS.
• the hybridization signal from hybridization reactions is detected.
• the detection may be carried out by various processes depending on a type of detectable labels linked with the NVS primers.
• the enzyme/substrate pair useful in this invention includes a pair of peroxidase ⁇ e.g., horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-metylarcridinium nitrate), resolupin benzyl ether, luminol, Amplex Red reagent (10-acethyl-3,7- dihydroxiphenoxazine), HYR (p-phenylenediamine-HCI and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2 ⁇ -Azine-di[3-ethylbenzthiazoline sulfonate]), o- phenylenediamine (OPD
• peroxidase ⁇ e.g., horseradish peroxidase
• chloronaphthol aminoethylcarbazole
• the detection may be carried out by the silver staining using silver nitrate.
• the step (d) comprises the sub-steps of:
• hybridization signals makes it possible to finally identify nucleotide variations in a nucleic acid molecule.
• the nucleic acid molecule is concluded to carry the homozygous genotype for the first nucleotide variation.
• the nucleic acid molecule is concluded to carry the homozygous genotype for the second nucleotide variation.
• the nucleic acid molecule is elucidated to contain both the first and second nucleotide variations (heterozygous genotype).
• the detection of hybridization signals may be carried out by a so-called "Array tube” technology known to one of skill in the art
• the present method is used to detect a wide variety of nucleotide variations.
• the present invention could be applicable in detection of a number of nucleotide variations in a human genome ⁇ e.g., variations in the MTHFR
• the present method is carried out to detect a drug-resistant pathogen, more preferably, a drug-resistant virus. It is preferred that the drug-resistant virus HIV (human immunodeficiency virus)-l, HIV-2,
• HBV hepatitis B virus
• HCV hepatitis C virus
• human herpesvirus most preferably HBV.
• drug-resistant viruses have resistance to at least one antiviral agent selected from the group consisting of zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delavirdine, efavirenz, adefovir, adefovir dipivoxil, FTC, D4FC, BCH-189, F-ddA, tetrahydroimidazo[4,5,l-jk]- [l,4]benzodiazepine-2(lH)-thione, (S)-4-isopropoxycarbonyl-6-methoxy-3-
• antiviral agent selected from the group consisting of zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delavirdine, efavirenz, adefovir, adefovir dipivoxil, FTC, D4FC, BCH-
• drug-resistant viruses have resistance to lamivudine.
• sequences of the NVS-P primers are designed with referring to mutated sequences of drug-resistant pathogens.
• Nucleotide variations in HIV-I reverse transcriptase conferring drug resistance to antiviral agents are exemplified as follows: Met41Leu, Glu44Asp, Glu44Ala, Ile50Val, Ala62Val, Lys65Arg, Asp67Asn, Ser68Gly, Thr69Asp, Thr69Ser-Ser-Gly, Thr69Ser-Thr-Gly, Thr69Ser-Val-Gly, Lys70Arg, Lys70Glu, Leu74Ile, Leu74Val, Val75Ile, Val75Leu, Val75Thr, Phe77Leu, LeulOOIIe, LyslO3Asn, VallO8Ala, VallO8Ile, Proll9Ser, Ilel35Thr, Ilel35Val, Glnl51Met, Thrl65Ile, Vall79As
• Nucleotide variations in HIV-I protease conferring drug resistance to antiviral agents are exemplified as follows: LeulOIIe, LeulOVal, LeulOPhe, Val32Ile, Glu35Asp, Met36Ile, Met46Ile, Met45Leu, Ile47Val, Gly48Val, Ile50Val, Ile54Met, Ile54Ser, Ile54Val, Leu63Pro, Ala71Thr, Ala71Val, Val77Ile, Val82Ala, Val82Ile, Val82Phe, Val82Thr, Ile84Ala, Ile84Val, Asn88Asp, Asn88Ser, Leu89Met, Leu89Pro and Leu90Met.
• Nucleotide variations in HCV RNA-dependent RNA polymerase conferring drug resistance to antiviral agents are exemplified as follows: Lys50Arg, Met71Val, Asn411Ser, Met414Thr, Phe415Ty and Val581Ala.
• Nucleotide variations in HCV NS5A protein conferring drug resistance to antiviral agents are exemplified as follows: Leu2190Lys, Val2198Leu, Val2198Met, Val2198Glu, Thr2217Ala, Thr2217Val, Asn2218Asp, Asn2218Lys, Asn2218Ser, Asp2220Glu, Asp2223Glu, Glu2225Asp, Glu2228Gln, Glu2236Ala, Asn2248Asp, He2252Val, Ile2268Val, Arg2276Leu, Lys2277Arg, Ser2278Pro, Arg2280Lys, Arg2280Glu, Ala2282Thr, Pro2283Gln, Pro2283Arg, Val2287Ile, Leu2298Val, Leu2298Ile, Thr2300Pro, Thr2300Ala, Lys2302Asn, Lys2303Asn, As
• Val555Ile adefovir dipivoxil: Asn236Thr.
• the sequences of the NVS-P primers used in this invention are designed with referring to mutated sequences of drug-resistant pathogens.
• the present method is applied to detect drug-resistant variations in HBV polymerase, more preferably lamivudine-resistant variations in HBV polymerase, most preferably drug-resistant variations in a 552nd codon of a gene of HBV polymerase.
• the present method is useful in detecting at least two nucleotide variations at a single site and/or different sites in a simultaneous manner.
• the term used herein "simultaneous" means that a single amplification reaction generates an amplified product to detect at least two nucleotide variations. Therefore, the present invention is essentially accompanied with multiplex amplifications. Where the present method is applied to detect nucleotide variations at a single site, it is very useful in detecting at least two SNPs in a simultaneous manner.
• the present method is applied to detect at least two nucleotide variations at different sites, i.e., adjacent sites or distant sites
• the following applications are illustrated: (i) a simultaneous detection of two nucleotide variations at adjacent sites in a single codon; and (ii) a simultaneous detection of at least two nucleotide variations at adjacent sites in different codons or at distant sites.
• the present method is very advantageous in detecting two nucleotide variations in a simultaneous manner.
• the NVS-Pl and NVS- P2 primers generally comprise an overlapping sequence with each other.
• primers having overlapping sequence are used in the same amplification reaction, a duplex between primers are formed to result in the generation of false amplification results.
• the NVS primers used in the present method overcomes the shortcomings associated with the formation of duplex structures, enabling to simultaneously detect two nucleotide variations.
• the present invention will be described in more detail with referring to examples of the present invention for detecting a lamivudine-resistant hepatitis B virus (HBV).
• HBV lamivudine-resistant hepatitis B virus
• nucleotide sequence coding for DNA polymerase of hepatitis B virus that causes a nucleotide variation to induce lamivudine resistance is obtained.
• the nucleotide sequence is publicly available, e.g., found in GenBank accession Nos. NC003977, AY167096, AY167095 and AY306136.
• the nucleotide sequence codes single base variations to induce lamivudine resistance
• the wild type sequence codes for methionine (YMDD motif)
• HBVs having resistance to antiviral agents such as lamivudine and famciclovir have substitutions in which the methionine residue in the YMDD motif is replaced by isoleucine (YIDD motif), valine (YVDD motif) or serine (YSDD motif).
• YIDD motif isoleucine
• YVDD motif valine
• YSDD motif serine
• g, a and tg nucleotides are mutated to t, g and gt nucleotides, respectively.
• the YVDD-R primer of SEQ ID N0:3 and the YIDD-F primer of SEQ ID N0:4 are used.
• the YVDD-R primer produces amplicons together with the HLT-F primer (SEQ ID NO:1) and the YIDD-F primer produces amplicons together with the HLT-R primer (SEQ ID NO:2).
• the HLT-F primer and the HLT-R primer are annealed to the outer region of variation- occurring regions and generate amplified products. These primers generate amplicons with predetermined sizes and the sizes of amplicons are different from each other.
• the target nucleotide sequences in samples are easily analyzed to have variations by observing the sizes of amplified products in electrophoresis.
• the NVS-P primers including the YVDD-R primer and the YIDD-F primer have overlapping sequences to be hybridized, they do not interfere with other primers in amplification reactions, leading to accuracy amplification results.
• the YSDD-F primer of SEQ ID NO:4 is used for detecting the YSDD motif. The present method is applied to simultaneously detect at least two nucleotide variations at distant sites.
• the present method is performed to simultaneously detect both a variation in the YIDD motif and a L528M variation that are located at distant sites in the gene of HBV DNA polymerase
• the YIDD-F primer of SEQ ID N0:4 and the L528M-R primer of SEQ ID NO: 6 are utilized.
• the L528M-R primer produces amplicons together with the HLT-F primer and the YIDD-F primer produces amplicons together with the the HLT-R primer.
• Detection methods for variations in Human genome namely, variation at 677 nd nucleotide in MTHFR gene, are represented by the following examples. According to the present invention, at least two nucleotide variations can be simultaneously identified in either quantitative or qualitative manner, without false results by a simple amplification reaction.
• the present invention is carried out in accordance with multiplex amplification using at least two primer set;
• the present invention enables to simultaneously identify at least two nucleotide variations in much higher specificity; (iii) the present invention exhibits excellent workability in multiplex amplifications, ensuring to detect at least two nucleotide variations in a single amplification reaction;
• the present invention enables to simultaneously detect at least two nucleotide variations at a single site, adjacent sites in a single codon or distant sites;
• the present invention enables to analyze nucleotide variations either quantitatively or qualitatively and shows a excellent applicability in automation.
• Fig. 1 schematically represents a process for detecting a nucleotide variation of human MTHER (methyenetetrahydrofolate reductase) gene in accordance with a specific embodiment of this invention.
• TSP target specific primer
• nucleotide variation specific primer was designed to successfully detect SNP in a PCR process by annealing specifically to 677C, labeled biotin at its 5'-end.
• the nucleotide variation specific primer used in this invention is prepared to have structures represented by the general formula I, resulting in hybridization with target sequences in higher specificity.
• primers having structures of the general formula I two specificity portions are physically and functionally separated by the separation portion, such that the overall hybridization specificity of primers is dually determined by the variation adjacent specificity portion and the variation specificity portion.
• nucleotide variation specific primer was designed to successfully detect SNP in a PCR process by annealing specifically to 677T, labeled biotin at its 5'- end.
• the symbol “I” denotes deoxyinosine in the sequence.
• the underlined “C” is a site to detect 677C.
• Genomic DNA was extracted from human blood by use of AccPrepTM Genomic DNA Extraction kit (Bioneer, South Korea) and used as a template.
• the multiplex PCR amplifications were conducted using 20 ⁇ l of reaction mixtures containing 2 ⁇ l of 10 x PCR reaction buffer (Roche) containing 15 m M MgCI 2 , 2 ⁇ l of dNTP (2 mM each dATP, dCTP, dGTP and dTTP), 20-30 ng of template DNA and 0.5 ⁇ l of Taq polymerase (5 units/ ⁇ l, Roche).
• the tube containing the reaction mixture was placed in a preheated (94 0 C) thermal cycler and the amplifications were then performed under the following thermal conditions: denaturation at 94 0 C for 15 min followed by 30-45 cycles of 94 0 C for 30 sec, 60-65 0 C for 1.5 min and 72°C for 1.5 min; followed by a 10-min final extension at 72°C.
• EXAMPLE IV Microarray Analysis First, a DNA microarray was fabricated. Amino-modified oligonucleotide probes were immobilized on the surface of a glass (3 x 3 mm in size) coated with epoxide. Two oligonucleotides serving as probes were designed. One of two probes was specifically bound to a first variation-specific PCR product, namely, specifically bound to amplified products of 677C genotype of the human MTHFR gene by use of a pair of TSPl and NVS-Pl. The other was specifically bound to a second variation-specific PCR product, namely, specifically bound to amplified products of 677T genotype of the human MTHFR gene by use of a pair of TSP2 and NVS-P2.
• the microarray was placed into ArryTubesTM (Clondiag Inc, Germany). Afterwards, the multiplex PCR products in Example III were hybridized with the probes in the ArrayTubes. For hybridization, the PCR products into the ArrayTubes were denaturated at 95°C for 5 min, incubated at 5O 0 C for 1 hour, and washed at 3O 0 C in 2 x SSC and 0.1% SDS.
• Streptavidin-HRP (horseradish peroxidase) was introduced into the ArrayTubes and incubated at 3O 0 C for 15 min.
• 100 ⁇ l of TMB (tetramethylbenzidine) substrate solution was added into the ArrayTubes and incubated for 10 min at room temperature. Then, the images of the ArrayTube were obtained by the ATR 03 reader (Clondiag Inc, Germany).
• the sample analyzed was determined as MTHFR gene 677C homozygous genotype. Where the color development was only observed for the probes specifically hybridizing with the second variation-specific PCR product, the sample analyzed was determined as MTHFR gene 677T homozygous genotype. In the case that the color development was observed for two types of the probes each specifically hybridizing with the first and second variation-specific PCR products, the sample analyzed was determined as MTHFR gene 677C/677T heterozygous genotype.
• the present invention allows to easily analyze (particularly, genotyping) DNA molecules in biological samples in a multiplex reaction manner without false analysis data.
• the present invention enables to simultaneously identify at least two nucleotide variations without false results by simple amplification and hybridization reactions, which avoids needs for restriction enzyme treatment and sequencing.

Landscapes

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

Applications Claiming Priority (2)

Publications (1)

Family

ID=39759658

Family Applications (1)

Country Status (2)

Cited By (3)

Citations (3)

• 2007
  • 2007-03-14 KR KR1020070024849A patent/KR100881924B1/ko active IP Right Grant
• 2008
  • 2008-02-25 WO PCT/KR2008/001086 patent/WO2008111741A1/en active Application Filing

Patent Citations (3)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events