WO2008051039A1

WO2008051039A1 - Method for detecting nucleotide variations

Info

Publication number: WO2008051039A1
Application number: PCT/KR2007/005291
Authority: WO
Inventors: Jong Yoon Chun
Original assignee: Seegene, Inc.
Priority date: 2006-10-25
Filing date: 2007-10-25
Publication date: 2008-05-02
Also published as: EP2084281A1; KR20080037128A; JP2010504752A

Abstract

The present invention relates to methods for detecting nucleotide variations. According to the present invention, at least two nucleotide variations in the target sequence can be accurately detected without false results by a simple amplification reaction without additional procedure such as restriction enzyme treatment and sequencing.

Description

METHOD FOR DETECTING NUCLEOTIDE VARIATIONS

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to methods for detecting nucleotide variations.

DESCRIPTION OF THE RELATED ART

A multitude of antibiotics, antimycotics and antiviral drugs have been developed since penicillin was found (Alexander Fleming, in 1928). With help of drug development activities, the human society becomes free of pathogen-infected diseases that are the main cause of human mortality. Therefore, the mortality due to infectious diseases is significantly reduced. However, pathogens showing drug- resistance is also increased. A recent representative includes methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and mutated AI viruses with resistant to oseltamivir phosphate (Tamiflu) that is the only therapeutics of avian influenza.

This drug resistance is ascribed to mutation in drug binding sites of drug- targeted biomolecules (mainly proteins) to result in weakened drug binding. Bacteria and fungi with multi-drug resistance are responsible for difficulties in therapeutic fields. Since biomolecules as drug targets are often plural, the studies and analysis for drug resistance are difficult. Unlikely, targets of drugs to viruses are relatively definite, the analysis for drug resistance of viruses have been performed without difficulty.

The approaches to overcome problems associated with drug resistance includes development of drug derivatives binding to mutated binding sites of drug targets or development of novel drugs acting on another targets.

The human conquer on pathogens by drug development, counteraction of pathogens to drug development activities by mutation and novel drug development for mutated pathogens have been continually undertaken. Among such continual events, one of the most important developments involves hepatitis B virus (HBV) and antiviral agents against it.

Three hundred and fifty million people are estimated to be infected with hepatitis B virus in the world. 15-40% of HBV-infected patients are at risk to be developed to chronic B hepatitis, liver cirrhosis and cancer in their lifetime. In this regard, the treatment aim to chronic B hepatitis is to inhibit HBV replication and development to other liver diseases. The approved therapeutics for B hepatitis include interferon, lamivudine and adefovir. Lamivudine, a nucleoside analogue, is a drug to inhibit reverse transcriptase activity of HBV DNA polymerase that catalyze the conversion of HBV pregenomic RNA to DNA sequences. This drug has been reported to exhibit plausible inhibitory effect on HBV replication and better histopathologic findings after administration, which is generally administered for chronic B hepatitis. Unfortunately, 14-32% of patients administered with lamivudine have been found to have YMDD motif mutation. The YMDD motif positioned in the C region of HBV DNA polymerase is mutated such that a Met residue at amino acid 552 is substituted with VaI (M552V) or He (M552I). The lamivudine sensitivity of the mutated form is 45-fold lower than that of the wild type target. The mutation incidence of the YMDD motif becomes increasing with getting longer lamivudine administration. In the event that the negative signal to HBV DNA after lamivudine treatment and then HBV DNA breakthrough showing positive signals to HBV DNA occurs, the YMDD mutation is very likely to generate.

To be free from shortcomings associated with the breakthrough of lamivudine resistant HPV, adefovir have been widely administered as substitution drugs. In past three-year clinical trials, about 3.9% of patients administered with adefovir were found to have adefovir-resistant HBV. This mutated HBV includes mutations at amino acid 236 (substitution of Asn wit Ala) in the D region and at amino acid 181 (substitution of Ala wit Thr) in the B region of HBV DNA polymerase. It has been known that the replication of the mutated HBV with adefovir resistance is inhibited by lamivudine. Therefore, the drug resistance to lamivudine or adefovir is measured by alternative administration of two drugs for preventing breakthrough of viral DNA molecules.

Because such mutations causing drugs resistance occur mostly in single or two nucleotides, their detection or identification is not ready. The recent methods for drug resistance viruses includes sequencing after PCR amplification, electrophoresis after restriction, mass analysis after restriction (PCR-RFMP), LightCycler probe hybridization, and primer-specific real-time PCR. The standardized kits commercially available comprise INNO-LiPA HBV DR (Innogenetics, Ghent, Belgium) for reverse hybridization and TRUGENE HBV genotyping kit (Visible Genetics/Bayer, Toronto, Canada) for directly nucleotide sequencing. Where viruses with different genotypes are mixed, more than 20% of total viruses have to comprise viruses with specific sequences for detecting viruses with specific sequences by sequencing methods. For detecting specific viruses by the LiPA method, more than 20% of total viruses have to comprise viruses with specific sequences.

The conventional methods described above have serious shortcomings such as longer assay time, intensive labor and lower accuracy. Accordingly, there remain needs to suggest novel approaches for detecting single-nucleotide differences in nucleic acid molecules such as genomic DNA in a much more convenient, accurate and sensitive fashions.

Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains. DETAILED DESCRIPTION OF THIS INVETNION

To meet long-felt needs in the art, 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.

Accordingly, it is an object of this invention to provide a method for simultaneously detecting at least two nucleotide variations of a target nucleotide sequence.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

In one aspect of this invention, there is provided 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) contacting the primers of the following (i)-(iv) to the target nucleotide sequence under a hybridization condition: (i) a first nucleotide variation specific primer 1 (NVS-Pl) hybridizable with a first variation-occurring region of the target nucleotide sequence having a nucleotide corresponding to a first nucleotide variation, wherein the NVS-Pl primer comprises a nucleotide complementary to the nucleotide corresponding to the first nucleotide variation; (ii) a target specific primer 1 (TSPl) 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; (iii) a second nucleotide variation specific primer 2 (NVS-P2) 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

(iv) 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; wherein a primer set comprising the primers (i) and (ii) and a primer set comprising the primers (iii) and (iv) are designed and prepared to generate amplification products with different sizes from each other, and the NVS-Pl and NVS-P2 primers are represented by the following general formula I:

wherein A_p represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the target nucleotide sequence, Y_q represents a separation portion comprising at least three universal bases, V₁- 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, and A, Y, and V are deoxyribonucleotide or ribonucleotide; T_m of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest T_m 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 specificity portion such that the overall hybridization specificity of the primers is enhanced; (b) performing at least two cycles of primer annealing, primer extending and denaturing using the primers to amplify the target sequence; and

(c) detecting the nucleotide variation by analyzing the sizes of the amplified products of the step (b).

The present invention is drawn to provide methods for simultaneously detecting various nucleotide variations or SNP (single nucleotide polymorphism) in target sequences. In particular, the present invention provides methods for simultaneously detecting nucleotide variations at the same (single) nucleotide or in the same (single) codon.

Up to now, it has not yet been proposed that at least two nucleotide variations, particularly at least two variations at the single nucleotide or in the single codon cannot be detected in a simultaneous manner solely by amplification reactions

{e.g., PCR). The present invention ensures first to suggest a practical method to simultaneously detect at least two variations by amplification reactions.

The term used herein "nucleotide variation" refers to a nucleotide polymorphism {e.g., SNP) in a wild type sequence. The nucleotide sequences to be detected by the present invention include gDNA, cDNA and RNA.

The fundamental structure of the primers used in this invention, particularly the nucleotide variation specific primers (NVS) has been first proposed by the present inventor and called as a structure with dual specificity. Therefore, oligonucleotides having such structure are named as dual specificity oligonucleotides

(DSO). The DSO embodies a novel concept and its hybridization is dually determined by the 5'-high T_m specificity portion and the 3'-low T_m specificity portion separated by the separation portion, exhibiting dramatically enhanced hybridization specificity (see

PCT/KR2005/001206).

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.

More specifically, 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. In this context, the NVS primers are annealed to target sequences in a different performance from conventional primers, which results in greatly increasing the annealing specificity.

According to a preferred embodiment, 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, PNA-5-introindole, PNA-nebularine, PNA- inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate- nebularine, phosphoramidate-inosine, phosphoramidate-4- nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethyl inosine, 2'0-methoxyethyl nebularine, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitrobenzimidazole, 2'-0-methoxyethyl 3-nitropyrrole, and combinations thereof. More preferably, the universal base or non-discriminatory base analog is deoxyinosine, 1- (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.

Preferably, 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.

It is preferable that the 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.

According to a preferred embodiment, the T_m of the variation adjacent specificity portion ranges from 40⁰C to 80⁰C, more preferably 45°C to 65°C. The T_m of the variation specificity portion ranges preferably from 10⁰C to 40⁰C. It is preferable that the T_m 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 3'-end of the variation specificity portion. More preferably, 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. Most preferably, 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. For instance, where the variation specificity portion is 8 nucleotides in length, 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.

Where the 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:

For example, according to the conventional methods for detecting SNP using the conventional primers, the nucleotide variation-specific base is located at the 3'- end of primers. This position is responsible for the slight difference in T_m values of annealing occurrence and non-annealing occurrence {i.e., mismatching) of the nucleotide variation-specific base. Thus, when mismatching events occur, amplification reactions are very likely to occur, giving rise to the generation of false results. Conversely, where the nucleotide variation-specific base is located around at the center of conventional primers, the difference in T_m values between annealing and non-annealing {i.e., mismatching) becomes larger. However, 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.

Surprisingly, the present invention ensures to completely overcome the shortcoming of conventional techniques described above. For illustration, where the nucleotide variation-specific base located around at the center of the variation specificity portion of the present primers is mismatched, the T_m value of the variation specificity portion becomes much lower because the mismatching occurs at the center of the variation specificity portion. However, considering the overall structure of the present primers, 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.

According to a preferred embodiment, the NVS primers used in this invention have the structure represented by the following general formula II:

5'-A_p-(dI)_q-V_r-3' (II) wherein A_p 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, V_r 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, and the nucleotide complementary or corresponding to the nucleotide variation is located at the center of V₁-.

In NVS primers of the general formula II, (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.

In NVS primers of the general formula II, the nucleotide complementary or corresponding to the nucleotide variation is located at the center of V₁-. For instance, where V_r 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, 4-6, 5-6, 5-7, 6-7 and 6-8 nucleotides apart from the 3'-end of V_r, respectively.

The term "primer" as used herein 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. Preferably, 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. For example, the primer used in this invention may include nucleotides with backbone modifications such as peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365:566-568(1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, nucleotides with sugar modifications such as 2'-O-methy! RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O- 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-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-), 7-deazapurines with C-7 substituents (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine, and diaminopurine.

The sequences of the primers may comprise some mismatches, so long as they can be hybridized with templates and serve as primers.

The term used "hybridizing" used herein 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. The process for amplifying the target nucleotide sequence by primer annealing, primer extending and denaturing is well known to those of skill in the art.

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). According to a preferred embodiment, the hybridization (annealing) is performed at temperature of 40-70⁰C, more preferably 45-68°C, still more preferably 50-65⁰C, most preferably 60-65⁰C.

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 80⁰C to 105⁰C. 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).

Where a mRNA is employed as starting material, 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, N.Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988)). For reverse transcription, 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).

A variety of 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. Preferably, 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. 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). Such stringent conditions for annealing will be sequence-dependent and varied depending on environmental parameters. Preferably, the annealing temperature ranges from 40⁰C to 70⁰C, more preferably from 45°C to 68⁰C, more preferably from 50⁰C to 65°C, most preferably from 6O⁰C to 65°C. In the most preferable embodiment, 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 analysis of amplified products in the present invention may be conducted by various methods or protocols, e.g. electrophoresis such as agarose gel electrophoresis. The primers used in the present invention are designed to produce amplicons with different sizes for identifying nucleotide variations; therefore the simple analysis or observation of size difference of amplified products enables to detect nucleotide variations in more convenient manner such as observation with naked eye.

Since 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. Preferably, 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.

According to a preferred embodiment, 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. For example, where the present method is performed to detect lamivudin-resistant hepatitis B virus (HBV), three NVS-P primers each hybridizable with YIDD motif, YVDD motif and L528M variations are used to simultaneously three types of variations.

According to a preferred embodiment, the step (a) is carried out using an additional primer pair to amplify a nucleotide sequence other than the target nucleotide sequence, whereby the step (a) additionally generates an internal control. The internal control is to verify the success of amplification reactions. For instance, as illustrated in Examples, the rbcL gene involved in photosynthesis of rice (Oryza sat/Va) is involved in amplification reactions as internal control and primers to amplify the rbcL gene are used. Primers for generating internal control may be designed to have conventional primer structures without the separation portion. Preferably, the primers have a structure represented by the general formula I without the nucleotide complementary or corresponding to the nucleotide variation.

According to a preferred embodiment, 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) or human herpesvirus, most preferably HBV.

According to a preferred embodiment, 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-

(methylthiomethyl)-3,4,-dihydroquinoxaline-2(lH)-thione, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, entecavir, famciclovir, benzo-l,2,4-thiadiazine antiviral agent, ribavirin and interferon. Most preferably, drug-resistant viruses have resistance to lamivudine.

According to the present method, the 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 {e.g., zalcitabine, zidovudine, didanosine and lamivudine) 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, VallOδAla, VallO8Ile, Proll9Ser, Ilel35Thr, Ilel35Val, Glnl51Met, Thrl65Ile, Vall79Asp, Tyrlδllle, Metl84Ala, Metl84Ile, Metl84Val, Tyrl88His, Tyrl88Leu, Glyl90Ala, Glyl90Cys, Glyl90Glu, Gryl90Gln, Glyl90Ser, Glyl90Thr, Leu210Trp, Leu214Phe, Thr215Tyr, Thr215Phe, Thr215Ser, Lys219Gln, Pro294Ser and Gly333Glu.

Nucleotide variations in HIV-I protease conferring drug resistance to antiviral agents {e.g., saquinavir, ritonavir, indinavir, nelfinavir and amprenavir) 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 {e.g., benzo-l,2,4-thiadiazine antiviral agent) are exemplified as follows: Lys50Arg, MerJlVal, Asn411Ser, Met414Thr, Phe415Ty and Val581Ala.

Nucleotide variations in HCV NS5A protein conferring drug resistance to antiviral agents {e.g., interferon) are exemplified as follows: Leu2190Lys, Val2198l_eu, 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, Asp2305Gly and Pro2315Ala.

Nucleotide variations in HBV polymerase conferring drug resistance to antiviral agents are exemplified as follows: (i) lamivudine: Leu426Ile, Leu426Val,

Vall73Leu, Met552Ile, Met552Val, Met552Ser, Val555Ile and Leu528Met; (ii) entecavir: Ilel69Thr, Thrl84Gly, Ser202Ile and Met250Val; (iii) famciclovir:

Vall73Leu, Met552Ile, Val555Ile; and (iv) 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.

According to a preferred embodiment, 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 552^nd 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. Where 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.

For detecting at least two nucleotide variations at a single site or at adjacent sites in a single codon, the present method is very advantageous in detecting two nucleotide variations in a simultaneous manner. In such a case, the NVS-Pl and

NVS-P2 primers generally comprise an overlapping sequence with each other.

Generally, where 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. However, 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).

First, the 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 single nucleotide polymorphisms conferring lamivudine resistance are indicated in Fig. 1. The wild type sequence codes for methionine (YMDD motif). However, 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). In the YIDD motif, YVDD motif and YSDD motif, g, a and tg nucleotides are mutated to t, g and gt nucleotides, respectively.

For simultaneously detecting the YVDD motif and YIDD motif, the YVDD-R primer of SEQ ID NO:3 and the YIDD-F primer of SEQ ID NO: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 (see Fig. 2). These primers generate amplicons with predetermined sizes and the sizes of amplicons are different from each other. Therefore, the target nucleotide sequences in samples are easily analyzed to have variations by observing the sizes of amplified products in electrophoresis. Although 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. For detecting the YSDD motif, the YSDD-F primer of SEQ ID NO:4 is used.

The present method is applied to simultaneously detect at least two nucleotide variations at distant sites. Where 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 NO: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.

According to the present invention, at least two nucleotide variations can be identified without false results by a simple amplification reaction. The features and advantages of this invention are will be summarized as follows:

(i) the present invention is carried out in accordance with multiplex amplification using at least two primer set; (ii) 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;

(iv) where the nucleotide complementary or corresponding to the nucleotide . variation in the variation specificity portion of the NVS-P primers is located at the center or around the center of the variation specificity portion, the single nucleotide- mismatch can differentiated in amplification reactions; (v) 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;

(vi) generally, where 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. In contrast, the present invention overcomes the shortcomings associated with the formation of duplex structures, preventing the generation of false results.

According to the present invention, at least two nucleotide variations in the target sequence can be accurately detected without false results by a simple amplification reaction without additional procedure such as restriction enzyme treatment and sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows various types of nucleotide variations causing lamivudine- resistance in polymerase of hepatitis B virus (HBV). Fig. 2 represents primers to detect HBV lamivudine resistance-causing nucleotide variations and their amplicon sizes.

Fig. 3a represents results of multiplex PCR amplifications according to the present invention for detecting SNPs in the CYP2C19 gene. Figs. 3b and 3c represents results of multiplex PCR amplifications using samples obtained from B hepatitis patients whose lamivudine resistance had been verified by DNA sequencing. Fig. 3b shows results of multiplex PCR using a primer annealing to outer region of variation-occurring regions, a YVDD variation specific primer and a YIDD variation specific primer. The Tightest lane and lane N represent 100 bp DNA marker and internal control, respectively. Lanes 1-2 represent patient samples infected with HBV having no lamivudine resistance. Lanes 3 and 4 represent patient samples infected with HBVs having YIDD variation and YIDD/L528M variations, respectively. Lanes 5 and 6 represent patient samples infected with HBVs having YVDD/L528M variations. Lanes 7 and 8 represent patient samples infected with HBVs having YIDD variation, YVDD variation and L528M variation.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

EXAMPLE I: Primer Design and Preparation EXAMPLE 1-1: Primers for Amplifying Nucleic Acids of Hepatitis B Virus

Based on the nucleotide sequence coding for DNA polymerase of Hepatitis B virus, a forward and reverse primers serving as TSP (target specific primer) were designed. TSP was designed to show sufficient PCR specificity even if it was designed according to conventional primer design. HLT-F 5'- CTC GTG GTG GAC TTC TCT CA -3' (SEQ ID NO: 1) HLT-R 5¹- GTG TAA AAG GGG CAG CAA AG -3¹ (SEQ ID NO:2)

The nucleotide sequence (781 bp) encoding HBV DNA polymerase was amplified using two primers. EXAMPLE 1-2: Primers for Amplifying Nucleic Acids of YVDD Lamivudine Resistant Hepatitis B Virus

Based on the nucleotide sequence of YVDD lamivudine-resistant hepatitis B virus, a nucleotide variation specific primer (NVS) was designed to successfully detect SNP in a PCR process by annealing specifically to YVDD variation-occurring site. YVDD-R 5'- CTT GGC CCC CAA TAC CAI III ITC CAC ATA -3' (SEQ ID NO: 3)

The symbol "I" denotes deoxyinosine in the sequence. The underlined "C" is a site to detect variation of YMDD→YVDD.

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. In 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. In addition, the sequence corresponding to or hybridizable with nucleotide variations is positioned in the middle of the variation specificity portion, whereby Tm differences in mismatch events become increasing and DNA synthesis by Taq polymerase does not occur in mismatch events.

EXAMPLE 1-3: Primers for Amplifying Nucleic Acids of YIDD Lamivudine Resistant Hepatitis B Virus

Based on the nucleotide sequence of YIDD lamivudine-resistant hepatitis B virus, a nucleotide variation specific primer (forward primer) was designed to successfully detect SNP in a PCR process by annealing specifically to YIDD variation-occurring site. YIDD-F 5'- CCC CAC TGT TTG GCT TTI III IAT ATT GAT -3" (SEQ ID NO:4) The symbol "I" denotes deoxyinosine in the sequence. The underlined "T" is a site to detect variation of YMDD→YIDD.

EXAMPLE 1-4: Primers for Amplifying Nucleic Acids of YSDD Lamivudine Resistant Hepatitis B Virus

Based on the nucleotide sequence of YSDD lamivudine-resistant hepatitis B virus, a nucleotide variation specific primer (forward primer) was designed to successfully detect SNP in a PCR process by annealing specifically to YSDD variation-occurring site. YSDD-F 5'- CCC CAC TGT TTG GCT TTI III IAT AGT GA -3¹ (SEQ ID NO:5)

The symbol "I" denotes deoxyinosine in the sequence. The underlined "GT" is a site to detect variation of YMDD→YSDD.

EXAMPLE 1-5: Primers for Amplifying Nucleic Acids of L528M Lamivudine Resistant Hepatitis B Virus

Based on the nucleotide sequence of L528M lamivudine-resistant hepatitis B virus, a nucleotide variation specific primer (reverse primer) was designed to successfully detect SNP in a PCR process by annealing specifically to YSDD variation-occurring site. L528M-R 5¹- AAC AAA TGG CGC TAG TAA III HG CCA TCA G -3' (SEQ ID NO: 6)

The symbol "I" denotes deoxyinosine in the sequence. The underlined "T" is a site to detect L528M variation.

EXAMPLE II: Preparation of Hepatitis B Viral DNA Molecules The nucleic acid molecules of hepatitis B virus were extracted using

AccuPrep™ Genomic DNA Extraction kit (Bioneer, South Korea) and used as templates for PCR amplifications.

EXAMPLE III: Internal Control In the rbcL gene involved in photosynthesis of rice {Oryza sativa), suitable sequences to be designed according to the general formula I were selected and used for designing a forward and revere primers. The symbol "I" denotes deoxyinosine. IC-F 5'- TAA ATC ACA GGC CGA AAC CGI III IAT TAA GGG GC -3' (SEQ ID NO: 7) IC-R 5¹- GTG AAT GTG AAG AAG TAG GCC GTT III HG GCA ATA ATG -3' (SEQ ID NO:8)

Using the rbcL gene sequence involved in photosynthesis of rice that is not found in human and human pathogens, the primers described above were prepared for internal control amplifications to verify the success of PCR amplifications in each PCR tube.

EXAMPLE IV: Multiplex PCR for Detecting Nucleic Acid Sequences of Laimivudine Resistant Hepatitis B Virus

The multiplex PCR amplifications were carried out using vial DNA samples prepared in Example II and primer pairs indicated in Tables 1 and 2. TABLE 1

The multiplex PCR amplifications were conducted using 20 μl of reaction mixtures containing 1 μl of template DNA, 2 μl of 10 x PCR reaction buffer (Roche) containing 15 mM MgCI₂, 2 μl of dNTP (2 mM each dATP, dCTP, dGTP and dTTP), 4 μl of 1.25 μM primer pair and 0.5 μl of ^ polymerase (5 units/μl, Roche). The tube containing the reaction mixture was placed in a preheated (94⁰C) thermal cycler and the amplifications were then performed under the following thermal conditions: denaturation at 94°C for 15 min followed by 30-45 cycles of 94°C for 30 sec, 60-65⁰C for 1.5 min and 72°C for 1.5 min; followed by a 10-min final extension at 72⁰C. PCR products were resolved by electrophoresis on an agarose gel containing EtBr (Figs. 3a-3b).

Fig. 3b represents results of multiplex PCR amplifications by use of three primer pairs including a primer annealing to outer region of variation-occurring regions, a YVDD variation specific primer and a YIDD variation specific primer described in Table 1. As shown in Fig. 3b, where DNA samples obtained from patients infected with HBV without lamivudine resistance were used for amplification, bands corresponding to amplification product (781 bp) by the primer annealing to outer region of variation-occurring regions and internal control product (205 bp) were only detected (lanes 1 and 2). Where DNA samples obtained from patients infected with YIDD variation HBV were used for amplification, the amplicon in 320 bp size was specifically produced with no false- negative and false-positive results by the YIDD variation specific primer (lane 3). Lane 4 corresponds to DNA samples obtained from patients infected with HBV having YIDD variation and L528M variation. The YIDD variation specific primer designed according to the present invention specifically generated the amplicon in 320 bp size with no false-negative and false-positive results. Where DNA samples obtained from patients infected with HBV having YVDD variation and L528M variation were used for amplification, the amplicon in 511 bp size was specifically produced with no false-negative and false-positive results by the YVDD variation specific primer (lanes 5 and 6). Lanes 7 and 8 correspond to DNA samples obtained from patients infected with HBV having three variations including YIDD variation, YVDD variation and L528M variation. The YIDD variation specific primer and YVDD variation specific primer designed according to the present invention specifically generated the amplicons in 320 bp size and 511 bp size, respectively, with no false-negative and false-positive results. Fig. 3c represents results of multiplex PCR amplifications by use of three primer pairs including a primer annealing to outer region of variation-occurring regions, a YVDD variation specific primer and a YIDD variation specific primer described in Table 2. As shown in Rg. 3c, where DNA samples obtained from patients infected with HBV without lamivudine resistance were used for amplification, bands corresponding to amplification product (781 bp) by the primer annealing to outer region of variation-occurring regions and internal control product (205 bp) were only detected (lanes 1 and 2). Where DNA samples obtained from patients infected with YIDD variation HBV were used for amplification, the amplification result was obtained with no false-negative and false-positive results (lane 3). Lane 4 corresponds to DNA samples obtained from patients infected with HBV having YIDD variation and L528M variation. The L528M variation specific primer designed according to the present invention specifically generated the amplicon in 442 bp size with no false-negative and false-positive results. Where DNA samples obtained from patients infected with HBV having YVDD variation and L528M variation were used for amplification, the amplicon in 442 bp size was specifically produced with no false-negative and false-positive results by the L528M variation specific primer (lanes 5 and 6). Lanes 7 and 8 correspond to DNA samples obtained from patients infected with HBV having three variations including YIDD variation, YVDD variation and L528M variation. The L528M variation specific primer designed according to the present invention specifically generated the amplicon in 442 bp size with no false-negative and false- positive results.

To summary, the results urge us to reason that the present invention ensures the specific detection of drug resistant genotypes of hepatitis B virus with no false-negative and false-positive results even under multiplex PCR conditions. In addition, it could be appreciated that the present invention enables not only to detect variations in a single nucleotide or single codon but also to simultaneously detect variations in adjacent positions {e.g., YIDD variation and L528M variation).

EXAMPLE V: Overlapping PCR Amplifications for Detecting SNP in the CYP2C19 Gene

Based on the nucleotide sequence coding for cytochrome P₄₅₀, subfamily HC (mephenytoin 4-hydroxylase), polypeptide 19 (CYP2C19) positioned in human chromosome 10q24, a forward and reverse primers serving as TSP (target specific primer) were designed.

CYP-TSP-F 5'-AGA GAA GAA TTG TTG TAA AAA GTA III HA TTA ATA TAA-3'

(SEQ ID NO:9) CYP-TSP-R 5'-AAA CTA GTC AAT GAA TCA CAA ATI III IAG CAG TCA C-3' (SEQ

ID NO:10)

Among SNP genotypes affecting the enzymatic activity of CYP2C19, nucleotide variation specific primers (NVS) were designed to successfully detect

SNPs in a PCR process by annealing specifically to allele 1 (681G) and allele 2A (681A).

NVS-Allele2-F 5'-TAA TTT TCC CAC TAT CAT TGA III IIT CCC AGG A-3' (SEQ ID NO:

H)

NVS-AIIeIeI-R 5'-CAA GGT TTT TAA GTA ATT TGT TII III TTC CCG GG-3' (SEQ ID NO: 12) The symbol "I" denotes deoxyinosine in the sequences. The underlined "C" and "A" are sites to detect allele 1 (681G) and allele 2A (681A) variations, respectively.

The TSP forward and reverse primers as well as the nucleotide variation specific primers used in this invention are prepared to have structures represented by the general formula I, resulting in hybridization with target sequences in higher specificity. In 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. In addition, the sequence corresponding to or hybridizable with nucleotide variations is positioned in the middle of the variation specificity portion, whereby Tm differences in mismatch events become increasing and DNA synthesis by Taq polymerase does not occur in mismatch events.

Multiplex amplification reactions were carried out using four primers mixed as indicated in Table 3. TABLE 3

The multiplex PCR amplifications were conducted using 20 μl of reaction mixtures containing 1 μl of template DNA, 2 μl of 10 x PCR reaction buffer (Roche) containing 15 mM MgCI₂, 2 μl of dNTP (2 mM each dATP, dCTP, dGTP and dTTP), 4 μl of 1.25 μM primer pair and 0.5 μl of Taq polymerase (5 units/μl, Roche). The tube containing the reaction mixture was placed in a preheated (94°C) thermal cycler and the amplifications were then performed under the following thermal conditions: denaturation at 94⁰C for 15 min followed by 30-45 cycles of 94°C for 30 sec, 60-65⁰C for 1.5 min and 72°C for 1.5 min; followed by a 10-min final extension at 72°C. PCR products were resolved by electrophoresis on an agarose gel containing EtBr (Figs. 3a). Fig. 3a represents results of multiplex PCR amplifications by use of three primer pairs including a primer annealing to outer region of variation-occurring regions, a allele 1 wile type specific primer and a allele 2 variation specific primer described in Table 3. As shown in Fig. 3a, where allele 1/allele 2 hetero DNA samples were used for amplification, all bands corresponding to amplification product (492 bp) by the primer annealing to outer region of variation-occurring regions, allele 1-specific product (321 bp) and allele 2-specific product (232 bp) were detected (lanes 1, 2 and 3). Where allele 1 homozygous DNA samples were used for amplification, bands corresponding to amplification product (492 bp) by the primer annealing to outer region of variation-occurring regions and allele 1- specific product (321 bp) were only detected (lanes 4, 5 and 6). Where allele 2 homozygous DNA samples were used for amplification, bands corresponding to amplification product (492 bp) by the primer annealing to outer region of variation- occurring regions and allele 2-specific product (232 bp) were only detected (lanes 7, 8 and 9). The allele 1 primer and allele 2 primer designed for differentiating single nucleotide variations according to the present invention specifically generated amplicons in 321 bp size ad 232 bp size, respectively, with no false- negative and false-positive results. However, the conventional method produced false results (see bottom panel of Fig. 3a).

To summary, the results lead us to reason that the present invention ensures the specific detection of single nucleotide polymorphism of human genes with no false-negative and false-positive results even under multiplex PCR conditions.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. 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) contacting the primers of the following (i)-(iv) to the target nucleotide sequence under a hybridization condition:

(i) a first nucleotide variation specific primer 1 (NVS-Pl) hybridizable with a first variation-occurring region of the target nucleotide sequence having a nucleotide corresponding to a first nucleotide variation, wherein the NVS-Pl primer comprises a nucleotide complementary to the nucleotide corresponding to the first nucleotide variation;

(ii) a target specific primer 1 (TSPl) 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; (iii) a second nucleotide variation specific primer 2 (NVS-P2) 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

5'-A_p-Y_q-V_r-3' (I) wherein A_p represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the target nucleotide sequence, Y_q represents a separation portion comprising at least three universal bases, V₁- 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, and A, Y, and V are deoxyribonucleotide or ribonucleotide; T_m of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest T_m 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 specificity portion such that the overall hybridization specificity of the primers is enhanced;

(b) performing at least two cycles of primer annealing, primer extending and denaturing using the primers to amplify the target sequence; and

2. The method according to claim 1, wherein the universal base is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2'-deoxyinosine, 2-aza-2'- deoxyinosine, 2'-OMe inosine, 2'-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2'- OMe 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, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4- nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino- nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3- nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4- nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethyl inosine, 2'0-methoxyethyl nebularine, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro- benzimidazole, 2'-0-methoxyethyl 3-nitropyrrole, and combinations thereof.

3. The method according to claim 2, wherein the universal base is deoxyinosine, 1- (2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole.

4. The method according to claim 1, wherein the variation adjacent specificity portion is 15-40 nucleotides in length.

5. The method according to claim 1, wherein the variation specificity portion is 3- 15 nucleotides in length.

6. The method according to claim 1, wherein the separation portion is 3-10 nucleotides in length.

7. The method according to claim 1, wherein the T_m of the variation adjacent specificity portion ranges from 40⁰C to 80⁰C.

8. The method according to claim 1, wherein the T_m of the variation specificity portion ranges from 10⁰C to 40⁰C.

9. The method according to claim 1, wherein the T_m of the separation portion ranges from 3°C to 15°C.

10. The method according to claim 1, wherein 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 3'-end of the variation specificity portion.

5

11. The method according to claim 10, wherein the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at 2-7 nucleotides apart from the 3'-end of the variation specificity portion. 0

12. The method according to claim 11, wherein the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at 3-6 nucleotides apart from the 3'-end of the variation specificity portion.

13. The method according to claim 10, wherein the nucleotide complementary or5 corresponding to the nucleotide variation in the variation specificity portion is located at the center of the variation specificity portion.

14. The method according to claim 1, wherein the target specific primers have a structure represented by the general formula I and the variation specificity portion0 has a nucleotide sequence substantially complementary to the target nucleotide sequence without the nucleotide complementary or corresponding to the nucleotide variation.

15. The method according to claim 1, wherein the variation specificity portions ofb the NVS-Pl and NVS-P2 primers comprise a partially overlapping sequence with each other.

16. The method according to claim 1, wherein 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.

17. The method according to claim 1, wherein the step (a) is carried out using an additional primer pair to amplify a nucleotide sequence other than the target nucleotide sequence, whereby the step (a) additionally generates an internal control.

18. The method according to claim 1, wherein the method is carried out to detect a drug-resistant pathogen.

19. The method according to claim 1, wherein the pathogen is HIV (human immunodeficiency virus)-l, HIV-2, HBV (hepatitis B virus), HCV (hepatitis C virus) or human herpesvirus.

20. The method according to claim 1, wherein the drug is 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-(methylthiomethyl)-3,4,- dihydroquinoxaline-2(lH)-thione, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, entecavir, famciclovir, benzo-l,2,4-thiadiazine antiviral agent, ribavirin and interferon.

21. The method according to claim 20, wherein the method is carried out for simultaneously detecting at least two nucleotide variations causing a lamivudine- resistance of HBV.

22. The method according to claim 21, wherein the nucleotide variation causing the lamivudine-resistance is a variation generated in a 552^nd codon of a gene of HBV polymerase.

23. The method according to claim 22, wherein the NVS-Pl or NVS-P2 primer is selected from the group consisting of SEQ ID NOs:3-6.