KR20080037128A - How to detect nucleotide variations - Google Patents

Abstract

The present invention relates to a method of simultaneously detecting at least two or more nucleotide variations of a target nucleotide sequence. According to the method of the present invention, two or more nucleotide variations can be accurately and simultaneously detected without errors through simple amplification reactions without conventional additional restriction enzyme processing or sequencing.

Description

Method for Detecting Nucleotide Mutations {Method for Detecting Nucleotide Variations}

Figure 1 shows the kind of nucleotide variation that causes lamivudine resistance in the polymerase of hepatitis B virus (HBV).

FIG. 2 shows the types of primers and the size of amplification products for detecting HBV lyvudine resistance-induced nucleotide variations, according to one specific embodiment of the invention.

Figure 3a shows the results of multiplex PCR for SNP detection in the CYP2C19 gene according to the method of the present invention.

Figure 3b and Figure 3c shows the results of multiplex PCR in accordance with the method of the present invention in a sample of hepatitis B patients confirmed the resistance to lamivudine by sequencing. FIG. 3b shows the results of multiplex PCR using primers annealed to the outer region of the region where mutations occurred, YVDD variant and YIDD variant specific primers, and a total of three primer pairs. The outermost lane is a 100 bp DNA molecular weight marker and lane N is the result of amplifying only the internal control. Lanes 1-2 are for samples of patients infected with HBV that are not lamivudine resistant. Lanes 3 and 4 are for samples of HBV-infected patients with YIDD variant and YIDD variant and L528M variant, respectively. Lanes 5 and 6 are for samples from HBV-infected patients with YVDD variant and L528M variant. Lanes 7 and 8 are for samples of HBV-infected patients with YIDD variant, YVDD variant, and L528M variant.

The present invention relates to a method for detecting nucleotide variations.

Since the discovery of penicillin (Alexander Fleming, 1928), humans have developed numerous types of antibiotics, antifungal agents, and antiviral agents. This has freed mankind from death and suffering from pathogen infections, which accounted for the largest part of human mortality. Thus, the mortality rate from pathogen infection is now extremely low. But pathogens also began to fight against humanity by increasing drug resistance as organisms. The emergence of mutation AI virus resistant to methicillin resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), or oseltamivir phosphate (Tamiflu), the only treatment for avian influenza, has recently become a social issue due to hospital infections. Yes.

Such drug resistance induces a mutation in the drug binding site of the biopolymer compound (mainly a protein) that is the target of the drug, causing problems in the drug binding. In the case of bacteria and fungi, multi-drug resistance can be induced, making treatment difficult. In addition, many biopolymers are not targeted, but many are not easy to analyze or study the drug resistance. Viruses, on the other hand, are easily analyzed for drug resistance because of the targeted drug targets.

The method of overcoming such drug-resistant pathogens is to develop a drug derivative that binds to a modified binding site of a drug target material to be used as a new drug or to develop a new drug for a new target material.

Hepatitis B virus is one of the most important factors for humankind, although the historical development of pathogens based on drug development, resistance to humans by pathogen mutations, and development of new drugs to conquer mutant strains are clear. (Hepatitis B virus, HBV) and its antiviral agents.

Approximately 350 million people in Korea and around the world are infected with the hepatitis B virus. Carriers of HBV have a high risk of developing chronic hepatitis B, cirrhosis and liver cancer in approximately 15-40% of their lives. Therefore, the purpose of treatment of chronic hepatitis B is to inhibit HBV replication and to prevent the progression of liver disease, and the currently recognized drugs for treatment of hepatitis B include interferon, lamivudine, and adefovir. .

Lamivudine is a drug that inhibits the reverse transcriptase function of HBV DNA polymerase when inserted into a viral DNA sequence which is reverse transcribed from the free genome RNA of HBV into a nucleoside analogue. It has been reported that HBV replication inhibitory effect is excellent and histologic findings are improved after treatment, and it is widely used as a treatment for chronic hepatitis B. However, 14-32% of patients receiving lamivudine for 1 year are known to develop YMDD motif variants. The YMDD motif is located in the C region of the HBV DNA polymerase gene. The YMDD motif is a site where the 552th Met of the amino acid sequence is replaced with Valine (M552V) or Ile (M552I) to generate a variant. The variant is more than 45 times less susceptible to lamivudine than the HBV wild type. The longer the treatment period of lamivudine, the higher the incidence of YMDD motif variants. Lamivudine therapy may be suspected of the appearance of YMDD variants when HBV DNA breakthrough occurs negatively in HBV DNA hybridization and then again with a very high level of positive response.

In the case of the appearance of lamivudine-resistant HBV, adefovir has been developed as an alternative medicine and is being used more and more. Drug-resistant viruses have been detected in approximately 3.9% of patients administered during the last three years of adefovir clinical trials. Molecularly, these viruses are mutated in HBV DNA polymerase region D (236) (Asn to Ala) and region B (181) (Ala to Thr). However, these adefovir resistant viruses are known to inhibit proliferation by lamivudine. Therefore, when drug resistance to lamivudine or adefovir occurs, a method of preventing the re-appearance of viral DNA by using two drugs alternately is used.

Since such drug resistance mutations are mostly caused by single or double nucleotide sequence changes, the detection method is not easy. At present, detection of drug-resistant mutant viruses is performed by sequencing the gene directly after PCR, electrophoresis after restriction enzyme treatment, mass spectrometry after restriction enzyme treatment (PCR-RFMP method), LightCycler probe hybridization, and primer-specific real. -time PCR method. Currently standardized kits are INNO-LiPA HBV DR (Innogenetics, Ghent, Belgium), which is detected by reverse hybridization, and TRUGENE HBV genotyping kit (Visible Genetics / Bayer, Toronto, which is detected by direct sequencing). Canada). If viruses with different genotypes are mixed, more than 20% of all viruses must be detected with a specific gene in order to detect viruses with a specific gene by sequencing. About 10% can be detected with LiPA method. Only a virus with a specific gene can be detected.

These methods have problems such as time consuming, excessive labor, and accuracy. Thus, there is a need for a simple, accurate and sensitive method of detecting single-base differences on nucleic acids such as genomic DNA.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors of the present invention have made a primer having a unique structure as a result of trying to solve the above-described needs of the art, when using this can not only accurately detect nucleotide variations without error, it is easy to simultaneously detect two kinds of variations This invention was completed by confirming that it can detect easily.

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

Other objects and advantages of the present invention will become apparent from the following examples and claims.

In accordance with an aspect of the present invention, the present invention provides a method for simultaneously detecting at least two or more nucleotide variations of a target nucleotide sequence in a sample comprising a target nucleotide sequence exhibiting a nucleotide variation comprising:

(a) contacting the primers of the following (i) to (iv) with the target nucleotide sequence under hybridization conditions:

       (i) a first nucleotide variant specific comprising a nucleotide that specifically hybridizes to a mutation-generating site of a target nucleotide sequence having a nucleotide corresponding to a first nucleotide variant and is complementary to the nucleotide corresponding to the first nucleotide variant Nucleotide variation specific primer 1: NVS-P1;

       (Ii) target specific primer 1: TSP1 that hybridizes to a specific site of a target nucleotide sequence located upstream of the mutation-occurring site where said NVS-P1 primer hybridizes;

       (Iii) specifically hybridizes to a sequence complementary to a mutation-occurring site of a target nucleotide sequence having a nucleotide corresponding to a second nucleotide variant at the same site, adjacent site or spaced apart site where the first nucleotide variant appears; A second nucleotide variant specific primer (NVS-P2) comprising a nucleotide corresponding to the second nucleotide variant; And

  (Iii) a target sequence specific primer 2: TSP2 that hybridizes to a specific site of a target nucleotide sequence located downstream of the mutation-producing site to which the NVS-P2 primer hybridizes, wherein (i) And the primer sets of (ii) and (iii) and (iii) primer sets are selected and designed to produce amplification products of different sizes, and the NVS-P1 and NVS-P2 primers are represented by the following general formula (I) Is displayed:

5'-A _p -Y _q -V _r -3 '(I)

In the general formula, A _p is a variation adjacent specificity portion having a hybridization sequence substantially complementary to the target sequence to be hybridized, and Y _q is a separation portion including at least three universal bases. V _r is a variation specificity portion comprising a nucleotide variant corresponding to or complementary to a nucleotide variant and having a hybridization sequence substantially complementary to the target sequence, p, q and r represent the number of nucleotides, X, Y and Z are deoxyribonucleotides or ribonucleotides, the Tm of the variant adjacent specificity zone is higher than the Tm of the variant specificity zone, the partitioning zone has the lowest Tm of the three zones, and the partitioning zone in terms of hybridization specificity Variation specificity zone Variation specificity zone Such specificity cleavage allows the hybridization specificity of the oligonucleotide overall structure to be determined double by the mutation adjacent specificity region and the variation specificity region, which in turn enhances the hybridization specificity of the oligonucleotide overall structure;

(b) performing at least two cycles of primer annealing, primer extension and denaturation to amplify the target nucleotide sequence; And,

(c) detecting nucleotide variations by confirming the size of the amplification products generated in step (b).

The present invention provides a method for simultaneously detecting various nucleotide variations or single nucleotide polymorphisms (SNPs) in a nucleotide sequence. In particular, the present invention provides a method capable of simultaneously detecting variations in the same base or in the same codon in a nucleotide sequence.

To date, no method has been developed that can simultaneously detect two or more nucleotide variations, in particular two or more variations in the same base or in the same codon, with a simple amplification reaction (eg, PCR). The present invention provides for the first time a practical method capable of confirming such simultaneous mutation detection with only a simple amplification reaction.

As used herein, the term "nucleotide variation" refers to a variation (eg, SNP) consisting of a change in the nucleotide of the wild type. The nucleotide sequence to be detected in the present invention means gDNA, cDNA and RNA.

The basic structure of the primers used in the present invention, in particular, nucleotide variation specific primer (NVS), is first proposed by the present inventors, may be referred to as a dual specificity structure, and has such a structure. Oligonucleotides are named dual specificity oligonucleotides (DSOs). DSO is a new concept oligonucleotide developed by the inventors, which is divided into 5'-high Tm specificity portions and 3'-low Tm specificity regions (3'-low) separated by a splitting region. Since hybridization is determined by Tm specificity portion, the hybridization specificity can be greatly improved (PCT / KR2005 / 001206).

NVS primers in the present invention is a modification of the DSO, and has the structure of Formula (I). NVS primers include a variation adjacent specificity portion, a separation portion and a variation specificity portion.

The partitioning region has the function of physically and functionally partitioning the variant adjacent specificity region and the variant specificity region. By such division, the variant adjacent specificity region and the variant specificity region are divided from each other in terms of hybridization specificity. This specificity cleavage allows the hybridization (annealing) specificity of the oligonucleotide overall structure to be determined double by the variant adjacent specificity region and the variant specificity region, which in turn enhances the hybridization specificity of the primer overall structure.

More specifically, when an NVS primer is annealed to a nucleotide variant-containing target sequence, the specificity is not determined by the full length of the primer as with conventional primers, but rather by the variant adjacent specificity region and the variant specificity region partitioned from each other. Is determined. Thus, when an NVS primer is annealed to a target sequence, it anneals in a different way from conventional primers, which results in greatly improved annealing specificity.

According to a preferred embodiment of the present invention, the universal base located in the division zone is deoxyinosine, inosine, 7-diaza-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, 1- (2'-di Oxy-beta-D-ribofuranosyl) -3-nitropyrrole, dioxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindol, di Oxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, dioxy 4-aminobenzimidazole, 4-aminobenzimidazole, dioxy nebulin, 2'-F nebulin, 2'-F 4 -Nitrobenzimidazole, PNA-5-introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, parent Lipolino-nebulin, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3- Tropirol, phosphoramidate-5-nitroindole, phosphoramidate-nebulin, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropy Roll, 2'-0-methoxyethylinosine, 2'-0-methoxyethyl nebulin, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro-benz Imidazole, 2'-0-methoxyethyl 3-nitropyrrole and combinations of said bases, more preferably dioxyinosine, inosine, 1- (2'-dioxy-beta- D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole, most preferably dioxyinosine.

According to a preferred embodiment of the invention, said partitioning zone comprises a continuous universal base.

Preferably, the length of the variant adjacent specificity region is longer than the variant specificity region. The variant contiguous specificity region preferably has a length of 15-40 nucleotides, more preferably 15-25 nucleotides in length.

The variant specificity region preferably has a length of 3-15 nucleotides, more preferably 5-15 nucleotides, most preferably 6-13 nucleotides in length.

The partitioning zone preferably has a length of 3-10 nucleotides, more preferably 4-8 nucleotides, most preferably 5-7 nucleotides.

According to a preferred embodiment of the invention, said variant adjacent specificity zone has a Tm of 40-80 ° C, preferably having a Tm of 45-65 ° C. Preferably, the variant specificity region has a Tm of 10-40 ° C. The partition zone preferably has a Tm of 3-15 ° C.

Variation specificity regions of the primers of the invention include nucleotide variations that correspond to or complement the nucleotide variations. When the primers of the present invention hybridize with the sense strand of the target nucleotide sequence, the variant specificity region includes nucleotides that are complementary to the nucleotide variant, and when hybridized with the antisense strand, include the nucleotide corresponding to the nucleotide variant.

According to a preferred embodiment of the present invention, the nucleotide corresponding to or complementary to the nucleotide variation is located at a position 1 to 10 bases away from the 3'-end or 3'-end of the mutation specific region, more preferably the mutation. Located 2 to 7 bases from the 3'-terminus of the specificity region, even more preferably 3 to 6 bases from the 3'-terminus. Most preferably, the nucleotide corresponding to or complementary to the nucleotide variant is located at or near the center of the variant specificity region. For example, where the mutation specific region comprises 8 nucleotides, the nucleotide corresponding to or complementary to the nucleotide variation is 3 to 6 bases apart from the 3'-terminus of the mutation specific region, preferably 4-5 bases. Are spaced apart, more preferably 4 bases apart.

In the mutation specific region of the NVS primers, if the nucleotide corresponding to or complementary to the nucleotide variation is located 3-6 bases apart or centered away from the 3′-end of the mutation specific region, Advantageous effects are produced.

For example, using a common primer, the mutation site is located at the 3'-end when detecting SNPs, in which case the base at the 3'-end is not annealed to the target sequence (i.e. In case of mismatching, Tm value is not very different. Therefore, even when mismatched, the amplification reaction is annealed and a false positive result tends to occur. Conversely, if the mutation site is placed in the center, most of the thermostable polymerizations differ slightly in the Tm value when the mutation site is annealed to the target sequence (ie when mismatched). The enzyme catalyzes the polymerization even in the mismatched part, leading to false positive results.

According to the present invention, the above-mentioned problems of the prior art can be completely overcome. For example, if a nucleotide variant corresponding or complementary nucleotide is located in the center of the variation specificity region, if mismatching occurs at this position, the mismatching occurs in the center of the structure only in the variation specificity region. However, the thermostable polymerase does not catalyze the polymerization reaction because the Tm value of the mutation specific region is greatly reduced compared with the match, and because of mismatching at the 3'-end in terms of the entire primer structure. Thus, if mismatched, no false positive result occurs.

According to a preferred embodiment of the present invention, the NVS primer used in the present invention is represented by the following general formula II:

5'-A _p- (dI) _q -V _r -3 '(I)

In the general formula, A _p is a variation adjacent specificity portion having a hybridization sequence substantially complementary to the target sequence to be hybridized, and (dI) _q is a partitioning region comprising consecutive deoxyinosine residues. portion, V _r is a variation specificity portion comprising a nucleotide variant corresponding to or complementary to a nucleotide variant and having a hybridization sequence substantially complementary to the target sequence, p is 15-25, q is 4 -8, r is 6-13, and the nucleotide corresponding to or complementary to the nucleotide variation is located in the center of V _r .

In the NVS primer of Formula II, (dI) is a division region comprising consecutive deoxyinosine residues, and the number of deoxyinosine residues is 4-8. In this case, other bases may be inserted within the range in which (dI) can serve as a division region.

In addition, in the NVS primer of Formula II, the nucleotide corresponding to or complementary to the nucleotide variation is located in the middle of V _r . For example, if V _r consists of 6, 7, 8, 9, 10, 11, 12, and 13 nucleotide residues, the nucleotides corresponding to or complementary to the nucleotide variation are each 3 from the 3'-terminus of V _r . It is preferred to be located at -4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7 and 6-8 nucleotides.

As used herein, the term "primer" means a synthetic or natural oligonucleotide. The primer serves as an initiation point for the synthesis under conditions in which the synthesis of the primer extension product complementary to the template is induced, i.e. the presence of polymers such as nucleotides and DNA polymerase, and conditions of suitable temperature and pH. For maximum efficiency of amplification, the primer is preferably single chain. Preferably, the primer is deoxyribonucleotide. Primers of the invention may include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include ribonucleotides. For example, the oligonucleotides of the present invention may be selected from the group consisting of backbone modified nucleotides such as peptide nucleic acids (PNA) (M. Egholm et al., Nature , 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexy Tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, Tityl-, propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro- , Iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazoryl-, pyridyl-), inosine and diaminopurine.

The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function.

The term "hybridization" as used herein means that two single stranded nucleic acids form a duplex structure by pairing of complementary base sequences. Hybridization can occur when perfect complementarity between single stranded nucleic acid sequences occurs or even when some mismatch base is present. The degree of complementarity required for hybridization may vary depending on the hybridization reaction conditions, and in particular, may be controlled by temperature. In general, if the hybridization temperature is high, there is a high probability that hybridization will occur in a perfect match, and if the hybridization temperature is low, hybridization may occur even if some mismatches are present. The lower the hybridization temperature, the higher the degree of mismatch that hybridization can occur.

Methods of amplifying target nucleotide sequences by carrying out primer annealing, primer extension and denaturation steps are well known in the art.

In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash time, buffer components and their pH and ionic strength depend on various factors such as the length and GC amount of the oligonucleotide and the target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization , Springer-Verlag New York Inc. NY (1999).

According to a preferred embodiment of the present invention, the hybridization (annealing) temperature is 40 ° C. to 70 ° C., more preferably 45 ° C. to 68 ° C., even more preferably 50 ° C. to 65 ° C., most preferably 60 ° C. To 65 ° C.

The method of the present invention for amplifying a nucleic acid sequence comprising a variant allows for detecting any desired variant. Such nucleic acid molecules are DNA or RNA. The nucleic acid molecule may be double stranded or single stranded, preferably double stranded. When the nucleic acid is a double strand as a starting material, it is preferable to make the two strands into a single strand or a partial single strand form. Methods for separating chains include, but are not limited to, heat, alkali, formamide, urea and glycoxal treatments, enzymatic methods (eg helicase action) and binding proteins. For example, chain separation can be accomplished by heat treatment to a temperature of 80-105 ° C. General methods of such treatments are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

If mRNA is used as the starting material, a reverse transcription step is required before amplification, and details of the reverse transcription step can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001). And Noonan, KF et al., Nucleic Acids Res. 16: 10366 (1988). In the reverse transcription step, oligonucleotide dT primers are used that hybridize to the poly A tail of the mRNA. Oligonucleotide dT primers consist of dTMPs, and one or more of the dTMPs can be replaced with other dNMPs, to the extent that the dT primer can act as a primer. The reverse transcription step can be accomplished by reverse transcriptase with RNase H activity. When using enzymes with RNase H activity, careful selection of reaction conditions can avoid individual RNase H cleavage.

Primers used in the present invention are hybridized or annealed to one site of the template to form a double chain structure. Conditions for nucleic acid hybridization suitable for forming such double chain structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

Various DNA polymerases can be used in the amplification step of the present invention and include "Clenau" fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu). Include. Most of the polymerases can be isolated from the bacteria themselves or can be purchased commercially. In addition, the polymerase used in the present invention can be obtained from cells expressing high levels of the cloning gene encoding the polymerase.

When carrying out the polymerization reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel. Excess of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. It is desired to provide cofactors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved.

All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

Annealing or hybridization in the present invention is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer (i.e., where the partitioning region cannot hydrogen bond to the target sequence). Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables. Preferably, the annealing temperature is 40 ° C to 70 ° C, more preferably 45 ° C to 68 ° C, even more preferably 50 ° C to 65 ° C, and most preferably 60 ° C to 65 ° C.

In the most preferred embodiment of the present invention, the amplification process is carried out according to the PCR disclosed in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.

Checking whether the amplification product is formed can be easily performed through electrophoresis, such as agarose gel electrophoresis. In particular, since the primers used in the present invention are designed so that the pairs have different amplification sizes from each other, the result of electrophoresis can be observed visually to easily determine whether nucleotide variations exist.

Since the target sequence specific primer (TSP) used in the present invention is annealed to the outer region where the mutation for analysis purposes occurs, a conventional primer, that is, a primer having a conventional primer structure without a division region, can be used. have. Preferably, the TSP has the structure represented by Formula I above, wherein the variant specificity region has a hybridization sequence that is substantially complementary to the target sequence without the nucleotide corresponding to or complementary to the nucleotide variant.

According to a preferred embodiment of the present invention, step (a) comprises one or more nucleotide variation specific primers that hybridize to other variants other than the hybridization of the NVS-P1 primer and the NVS-P2 primer. It is additionally used. For example, when applying the method of the present invention to detect lamivudine resistant variants of hepatitis B virus (HBV), it is possible to use three or more NVS-P primers that hybridize to YIDD motifs, YVDD motifs and L528M mutations. Three variations can be detected simultaneously.

According to a preferred embodiment of the present invention, step (a) is carried out by additionally using a pair of primers to generate an internal control by amplifying a nucleotide sequence other than the target nucleotide sequence of the mutation detection target. This internal control allows you to check that the amplification reaction itself is successful. For example, as illustrated in the following examples, for use as an internal control of the amplification reaction in addition to the HBV genome sequence of the mutation detection target, rbcL gene, which is a photosynthesis related gene of rice ( Oryza sativa ), is added to the amplification reaction. The primer for amplifying this rbcL gene is used. The primer for generating the internal control may have a conventional primer structure, but preferably has the structure of Formula (I). In this case, the variant specificity region of Formula I has a hybridization sequence that is substantially complementary to the target sequence without the nucleotide corresponding to or complementary to the nucleotide variant.

According to a preferred embodiment of the present invention, the method of the present invention is for detecting drug resistant pathogens, more preferably drug resistant viruses. The drug resistant virus is preferably human immunodeficiency virus (HIV) -1, HIV-2, hepatitis B virus (HBV), hepatitis C virus (HCV) or human herpesvirus (HBV), and most preferably HBV. to be.

According to a preferred embodiment of the present invention, the medicament to which the virus is resistant is zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, Delavirdine, efavirenz, adefovir, adefovir dipivoxil, FTC, D4FC, BCH-189, F-ddA, tetrahydroimidazo [4, 5,1-jk [] 1,4] benzodiazepine-2 (1H) -one (tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -one), tetrahydroimidazo [ 4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -thione (tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -thione), (S) 4-isopropoxycarbonyl-6-methoxy-3- (methylthiomethyl) -3,4-dihydroquinoxalin-2 (1H) -thione ((S) -4-isopropoxycarbonyl-6-methoxy- 3- (methylthiomethyl) -3,4, -dihydroquinoxaline-2 (1H) -thione), saquinavir, ritonavir, indinavir, nelfina Nelfinavir amprenavir, entecavir, famciclovir, benzo-1,2,4-thiazine antiviral agent, At least one antiviral agent selected from the group consisting of ribavirin and interferon, most preferably lamivudine.

According to the method of the present invention, an NVS-P primer is prepared by referring to the nucleotide sequence of the pathogen that exhibits drug resistance.

Examples of mutations in HIV-1 reverse transcriptase that result in resistance to antiviral agents (such as zalcitabine, zidovudine, didanosine, lamivudine, etc.) are 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, LeslOlIle, Vall , Ilel35Val, Glnl51Met, Thrl65Ile, Vall79Asp, Tyrl81Ile, Metl84Ala, Metl84Ile, Metl84Val, Tyrl88His, Tyrl88Leu, Glyl90Ala, Glyl90Cys, Glyl90Ser, Glyl90Trr, Lyu210Tr 215

Examples of mutations that occur in HIV-1 proteases that indicate resistance to antiviral agents (saquinavir, ritonavir, indinavir, nlpinavir, amprenavir, etc.) include: Leu10Ile, LeulOVal, LeulOPhe, Val32Ile, Glu35Asp, Met36Ile, Met46Ile, Met45Leu, Ile47Val, Gly48Val, Ile50Val, Ile54Met, Ile54Ser, Ile54Val, Leu63Pro, Ala71Thr, Ala71Val, Val77Ile, Val82AlaTalPheI, Pal As Phe 82 Leu89Pro and Leu90Met

Examples of variations in HCV RNA-dependent RNA polymerase that result in resistance to antiviral agents (eg, benzo-1,2,4-thiazine antiviral agents) include: Lys50Arg, Met71Val, Asn41 lSer, Met414Thr, Phe415Ty and Val581Ala.

Examples of mutations in the HCV NS5A protein resulting in resistance to antiviral agents (eg, interferon, etc.) include: Leu2190Lys, Val2198Leu, Val2198Met, Val2198Glu, Thr2217Ala, Thr2217Val, Asn2218Asp, Asn2218Lys, Assp2218220Ger, Asp218220Ger, Asp218220Ger, Asp218220 , Glu2225Asp, Glu2228Gln, Glu2236Ala, Asn2248Asp, He2252Val, Ile2268Val, Arg2276Leu, Lys2277Arg, Ser2278Pro, Arg2280Lys, Arg2280Glu, Ala2282Thr, Pro2283Gln, Pro2283Arg, Val2287Ile, Leu2298Val, Leu2298Ile, Thr2300Pro, Thr2300Ala, Lys2302Asn, Lys2303Asn, Asp2305Gly and Pro2315Ala.

Examples in which mutations in HBV polymerase occur to show resistance to antiviral agents are as follows: (iii) Lamivudine: Leu426Ile, Leu426Val, Val173Leu, Met552Ile, Met552Val, Met552Ser, Val555Ile, Leu528Met; (Ii) entecavir: Ile169Thr, Thr184Gly, Ser202Ile, Met250Val; (Iii) famciclovir: Val173 Leu, Met552Ile, Val555Ile; And (iii) adefovir difficile: Asn236Thr.

Nucleotide variant specific NVS-P primers used in the present invention are designed with reference to the mutated nucleotide sequences of the drug resistant pathogens described above.

According to a preferred embodiment of the invention, the invention is directed to a drug resistant nucleotide variant in a polymerase of HBV, more preferably a lamivudine resistant nucleotide variant in a polymerase of HBV, most preferably at 552th in a polymerase of HBV. Applied to detect variations in codons.

The present invention is useful for simultaneously detecting two or more nucleotide variations occurring at the same site and / or at different sites. As used herein, the term "simultaneously" means to obtain a reaction result that can confirm the presence or absence of two or more mutations in one amplification reaction solution. Therefore, according to the method of the present invention, the multiplex amplification reaction is necessarily accompanied.

When the method of the present invention is used to detect mutations at the same site, it is useful for detecting two SNPs simultaneously. When the method of the present invention is used to detect two or more kinds of mutations occurring at different sites, that is, adjacent or spaced sites, there are largely the following applicability. (Iii) simultaneously detect two nucleotide variations at adjacent sites in the same codon but not at the same site; And (ii) simultaneously detecting two or more nucleotide variations at adjacent sites or spaced apart locations that are not included in the same codon.

In the case of detecting two or more mutations in the same site or in the same codon, the present invention is advantageous for detecting two kinds of nucleotides simultaneously. In this case, the NVS-P1 primer and the NVS-P2 primer to be used generally have overlapping sequences, and may hybridize with each other to form a duplex. Dimer formation between these primers can make the amplification abnormal, resulting in false amplification results. However, the NVS primer of the present invention overcomes the problems caused by the formation of dimers between the primers, and this feature and advantage make it possible to detect two variations simultaneously.

According to one specific embodiment of the present invention for detecting lamivudine resistant variants of hepatitis B virus (HBV), the process of the present invention will be described as follows.

First, the coding sequence of the DNA polymerase of HBV as a target sequence from which nucleotide variations inducing lamivudine resistance occurs is obtained. Such sequences are known in the art and can be found, for example, in GenBank Accession Numbers NC003977, AY167096, AY167095 and AY306136. The single base mutation that causes lamivudine resistance in this sequence is shown in FIG. 1, and in the wild type it encodes methionine (YMDD motif). However, HBV resistant to antiviral agents such as lamivudine and famcyclovir have mutations in the YMDD motif where methionine is replaced with isoleucine (YIDD motif), valine (YVDD motif) or serine (YSDD motif). In the YIDD motif, g is changed to t, in the YVDD motif, a is converted to g, and in the YSDD motif, tg is changed to gt.

In order to detect variant YVDD motifs and YIDD motifs simultaneously, the YVDD-R primers of SEQ ID NO: 3 and the YIDD-F primers of SEQ ID NO: 4 are used. YVDD-R primers pair with HLT-F primers (SEQ ID NO: 1) to form amplification products, and YIDD-F primers pair with HLT-R primers (SEQ ID NO: 2) to form amplification products do. HLT-F and HLT-R are primers that anneal to the outer region where mutations occur, and these two primers may be paired with each other to form an amplification product (see FIG. 2). The primers are selected and designed such that the amplification products produced by them have a predetermined size, and the sizes of the amplification products produced by each primer set are different from each other. Therefore, by checking only the size of the amplification product through simple electrophoresis, it is possible to easily detect what mutation the target nucleotide sequence in the sample has. Although YVDD-R and YIDD-F corresponding to NVS-P primers have sequences that can hybridize with each other, they do not affect each other in the amplification reaction and show accurate amplification results. When the YSDD motif is to be detected, the YSDD-F primer of SEQ ID NO: 4 may be used.

The method of the present invention can be usefully used to simultaneously detect two or more variations in positions spaced from each other. If the method of the present invention is applied to simultaneously detect YIDD-F motifs and L528M mutations at mutually spaced positions on the HBV DNA polymerase gene, the YIDD-F primer and SEQ ID NO: 6 of SEQ ID NO: 4 L528M-R primers of the sequence are used. L528M-R pairs with HLT-F primers to form amplification products, and YIDD-F primers pair with HLT-R primers to form amplification products.

According to the method of the present invention, through a simple amplification reaction, it is possible to accurately detect two or more nucleotide variations simultaneously without errors.

In summary, the features and advantages of the present invention are as follows:

(Iii) The method of the present invention is carried out according to a multiplex amplification reaction using at least two or more primer sets.

(Ii) According to the method of the present invention, two or more nucleotide variations on the target nucleotide sequence can be detected simultaneously with very high specificity.

(Iii) The method of the present invention exhibits very good workability in multiplex reactions so that two or more nucleotide variations can be detected simultaneously in one amplification reaction set.

(Iii) In the NVS-P primer used in the method of the present invention, if a nucleotide variation or complementary nucleotide is located in the center of the variation specificity region, a single base mismatch can be completely distinguished from the amplification reaction. have.

(Iii) According to the method of the present invention, not only nucleotides in the same site or in the same codon, but also two or more nucleotides in spaced positions can be detected simultaneously.

(Iii) In general, when primer pairs having overlapping sequences are used, the primers may form dimers, resulting in an incorrect amplification. According to the method of the present invention, it is possible to overcome this problem of dimer formation. Amplification results can be prevented.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Example I: Primer Design and Preparation

Example I-1: Primer for Hepatitis B Virus Nucleic Acid Amplification

Based on the nucleotide sequence encoding the DNA polymerase of hepatitis B virus, forward and reverse primers were designed that can serve as target specific primers (TSPs). TSP was designed by selecting a sequence capable of exhibiting sufficient PCR specificity, even if designed according to the conventional primer design method as possible.

HLT-F 5'- CTC GTG GTG GAC TTC TCT CA -3 '(SEQ ID NO: 1)

HLT-R 5'- GTG TAA AAG GGG CAG CAA AG -3 '(SEQ ID NO: 2)

By these two primers, 781 bp HBV DNA polymerase-encoding sequence is amplified.

Example I-2 Primer for Amplifying YVDD Type Lamivudine Resistant Hepatitis B Virus Nucleic Acid

Based on the YVDD type lamivudine resistant hepatitis B virus nucleic acid sequence, a nucleotide variation specific primer (NVS) that can be specifically annealed at the site of YVDD mutation to successfully detect SNPs according to PCR method. Designed.

YVDD-R 5'- CTT GGC CCC CAA TAC CAI III ITC CA C ATA -3 '(SEQ ID NO: 3)

I in the sequence represents deoxyinosine. The underlined C in the sequence is the site for detecting YMDD-> YVDD variation.

Nucleotide variant specific primers used in the present invention have a structure of general formula (I), so that the hybridization with the target sequence with a very high specificity. In these primers having a structure of Formula I, the two specificity regions are physically and functionally divided by the cleavage region, and the hybridization specificity of the entire primer structure is dually regulated by the mutation adjacent specificity region and the variation specificity region. In addition, the base corresponding to the nucleotide variation or hybridization to the mutation is located in the central portion of the mutation specific region, so as to increase the Tm value according to the mismatch while preventing the DNA synthesis by Taq polymerase during mismatch.

Example I-3 Primer for Amplifying YIDD Type Lamivudine Resistant Hepatitis B Virus Nucleic Acid

Based on the YIDD type lamivudine resistant hepatitis B virus nucleic acid sequence, a nucleotide variant specific primer (forward primer) was designed that was specifically annealed to the site of the YIDD mutation to successfully detect SNPs according to PCR method. .

YIDD-F 5'- CCC CAC TGT TTG GCT TTI III IAT AT T GAT -3 '(SEQ ID NO: 4)

 I in the sequence represents deoxyinosine. The underlined T in the sequence is the site for detecting YMDD-> YIDD mutations.

Example I-4 Primer for Amplifying YSDD Type Lamivudine Resistant Hepatitis B Virus Nucleic Acid

Based on the YSDD type lamivudine resistant hepatitis B virus nucleic acid sequence, a nucleotide variant specific primer (forward primer) was designed that was specifically annealed to the site of YSDD mutation to successfully detect SNPs according to PCR method. .

YSDD-F 5'- CCC CAC TGT TTG GCT TTI III IAT A GT GA -3 '(SEQ ID NO: 5)

I in the sequence represents deoxyinosine. The underlined GT in the sequence is the site for detecting YMDD-> YSDD variations.

Example I-5: Primer for Amplifying L528M Type Lamivudine Resistant Hepatitis B Virus Nucleic Acid

Based on the L528M type lamivudine resistant hepatitis B virus nucleic acid sequence, a nucleotide variant specific primer (reverse primer) was designed that was specifically annealed to the L528M mutation site to successfully perform SNP detection according to the PCR method.

L528M-R 5'- AAC AAA TGG CGC TAG TAA III IIG CCA T GA G -3 '(SEQ ID NO: 6)

I in the sequence represents deoxyinosine. The underlined T in the sequence is the site for detecting the L528M mutation.

Example II Preparation of Hepatitis B Virus DNA

AccuPrep ^™ nucleic acid is used to detect hepatitis B virus nucleic acid using blood from a person infected with hepatitis B virus. Genomic DNA Extraction Kit (Bioneer, South Korea) was used to extract and used as a template for PCR amplification.

Example III Internal Control

Forward and reverse primers were designed using sequences suitable for designing the primers of Formula I, among the sequences discovered in rbcL gene sequence, a photosynthesis related gene of rice ( Oryza sativa ), for use as an internal control of PCR reaction. I in the sequence represents deoxyinosine.

IC-F 5'- TAA ATC ACA GGC CGA AAC CGI III IAT TAA GGG GC -3 '(SEQ ID NO: 7)

IC-R 5'- GTG AAT GTG AAG AAG TAG GCC GTT III IIG GCA ATA ATG -3 '(SEQ ID NO: 8)

The primers were prepared for amplification of the internal control group for confirming the success of PCR in each PCR tube using rbcL gene of rice photosynthesis related to no human or human pathogen.

Example IV Multiplex PCR to Detect Nucleic Acid Sequences of Lamivudine Resistant Hepatitis B Virus

Multiplex PCR was performed using the viral DNA samples obtained in Example II, respectively, using the primer pairs shown in Tables 1 and 2 below.

target Primer pair PCR product size (bp) HBV DNA Polymerase Gene HLT-F / HLT-R 781 YVDD Mutant Virus HLT-F / YVDD-R 511 YIDD Mutant Virus YIDD-F / HLT-R 320 Internal Control IC-F / IC-R 205

target Primer pair PCR product size (bp) HBV DNA Polymerase Gene HLT-F / HLT-R 781 L528M Mutant Virus HLT-F / L528M-R 442 YSDD variant virus YSDD-F / HLT-R 320 Internal Control IC-F / IC-R 205

2 μl of 10 × PCR reaction buffer (Roche) containing 15 mM MgCl ₂ , 4 μl of 1.25 μM primer pair, 2 μl of dNTP (2 mM dATP, dCTP, dGTP and dTTP, respectively), 1 μl and 0.5 μl of template DNA Multiplex PCR was performed using a final 20 μl reaction mixture containing Taq polymerase (5 units / μl, Roche). The tube containing the reaction mixture was placed in a preheated (94 ° C.) thermal cycler and denatured at 94 ° C. 15 minutes, followed by 94 ° C. 30 seconds, 60-65 ° C. 1.5 minutes, and 72 ° C. 1.5 minutes. 30-45 cycles of and then reacted at 72 ° C for 10 minutes. PCR products were electrophoresed on agarose gels containing EtBr, and the bands shown were identified (FIGS. 3A-3B).

FIG. 3B shows multiplex PCR using primers annealed to the outer region of the region in which the mutations described in Table 1 occurred, YVDD variant and YIDD variant specific primers, and a total of three primer pairs. Show results. As shown in FIG. 3B, in samples of patients infected with lamivudine-resistant HBV, amplifiers (781 bp) and internal control amplification produced by primers annealed to the outer region of the site where the mutation occurred Only bands corresponding to water (205 bp) were observed (lanes 1 and 2). Lane 3 is for a sample of an HBV-infected patient with YIDD variant. The 320 bp amplification product was correctly detected by the YIDD variant primer designed in this experiment, and no false positive and false negative results were observed. Lane 4 is for a sample of HBV-infected patients with YIDD variant and L528M variant, where 320 bp amplification products were correctly detected by the YIDD variant primers designed in this experiment, and no false positive and false negative results were observed. . Lanes 5 and 6 were for samples from HBV-infected patients with YVDD and L528M variants, with the 511 bp amplification product accurately detected by the YVDD variant primers designed in this experiment, and false-positive and false-negative results were observed. It wasn't. Lanes 7 and 8 are for samples from HBV-infected patients with YIDD variant, YVDD variant, and L528M variant, 320 bp and 511 bp, respectively, by the YIDD variant primers and YVDD variant primers designed in this experiment. The amplification product was detected correctly and no false positive and false negative results were observed.

FIG. 3C shows multiplex PCR using primers annealed to the outer region of the region in which the mutations described in Table 2 occurred, YSDD variant and L528M variant specific primers, and a total of three primer pairs. Show results. As can be seen in Figure 3c, in samples of patients infected with lamivudine-resistant HBV, amplifiers (781 bp) and internal control amplification produced by primers annealed to the outer region of the site where the mutation occurred Only bands corresponding to water (205 bp) were observed (lanes 1 and 2). Lane 3 is for samples from HBV-infected patients with the YIDD variant, where no false positive and false negative results were observed. Lane 4 is for a sample of HBV-infected patients with YIDD variant and L528M variant. The 528 bp amplification product was correctly detected by the L528M variant primer designed in this experiment, and no false positive and false negative results were observed. . Lanes 5 and 6 are for samples of HBV-infected patients with YVDD and L528M variants, where the 442 bp amplification product was correctly detected by the L528M variant primers designed in this experiment, and false-positive and false-negative results were observed. It wasn't. Lanes 7 and 8 are for samples from HBV-infected patients with YIDD variant, YVDD variant and L528M variant, where the 442 bp amplification product was correctly detected by the L528M variant primer designed in this experiment. No false negative results were observed.

In summary, it can be seen that according to the method of the present invention, drug resistance genotypes of hepatitis B virus can be accurately identified even under conditions of multiplex PCR without false-positive and false-negative errors. In addition, it can be seen that, according to the method of the present invention, not only gene mutations occurred at the same base or in the same codon, but also adjacent gene variants (eg, YIDD variant and L528M variant) can be detected at the same time. have.

Example V: Overlap PCR to Detect SNP in CYP2C19 Gene

Based on nucleotide sequences encoding cytochrome P450, a subfamily IIC (mephenytoin 4-hydroxylase), and polypeptide 19 (CYP2C19), genes located on human chromosome 10q24, can serve as target specific primers (TSPs). Forward and reverse primers were designed.

CYP-TSP-F 5'-AGA GAA GAA TTG TTG TAA AAA GTA III IIA 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 the SNP genotypes affecting the enzyme activity of CYP2C19, nucleotide variation specific primers can be annealed specifically to allele 1 (681G) and allele 2A (681A) to successfully detect SNPs by PCR method. : NVS) was designed.

NVS-Allele2-F 5'-TAA TTT TCC CAC TAT CAT TGA III IIT CCC A GG A-3 '(SEQ ID NO: 11)

NVS-Allele1-R 5'-CAA GGT TTT TAA GTA ATT TGT TII III TTC C C G GG-3 '(SEQ ID NO: 12)

I in the sequence represents deoxyinosine. Underlined C and A in the sequence are sites for detecting allele 1 (681G) and allele 2 (681A) mutations.

The TSP forward and reverse primers and nucleotide variant specific primers used in the present invention basically have the structure of general formula I, so as to hybridize with the target sequence with very high specificity. In these primers having a structure of Formula I, the two specificity regions are physically and functionally divided by the cleavage region, and the hybridization specificity of the entire primer structure is dually regulated by the mutation adjacent specificity region and the variation specificity region. In addition, the base corresponding to the nucleotide variation or hybridization to the mutation is located in the central portion of the mutation specific region, so as to increase the Tm value according to the mismatch while preventing the DNA synthesis by Taq polymerase during mismatch.

Multiplex PCR was performed by mixing all four primers as shown in Table 3.

target Primer pair PCR product size (bp) CYP2C19 Control Gene CYP-TSP-F / CYP-TSP-R 492 allele 1 (681G) CYP-TSP-F / NVS-Allele1-R 321 allele 2 (681A) NVS-Allele2-F / CYP-TSP-R 232

2 μl of 10 × PCR reaction buffer (Roche) containing 15 mM MgCl ₂ , 4 μl of 1.25 μM primer pair, 2 μl of dNTP (2 mM dATP, dCTP, dGTP and dTTP, respectively), 1 μl and 0.5 μl of template DNA Multiplex PCR was performed using a final 20 μl reaction mixture containing Taq polymerase (5 units / μl, Roche). The tube containing the reaction mixture was placed in a preheated (94 ° C.) thermal cycler and denatured at 94 ° C. 15 minutes, followed by 94 ° C. 30 seconds, 60-65 ° C. 1.5 minutes, and 72 ° C. 1.5 minutes. 30-45 cycles of and then reacted at 72 ° C for 10 minutes. PCR products were electrophoresed on agarose gels containing EtBr, and the bands shown were identified (FIG. 3A).

FIG. 3A shows multiplex PCR using primers annealed to the outer region of the region in which the mutations described in Table 3 occurred, allele 1 wild type and allele 2 variant specific primers, and a total of three primer pairs. Show the results. As can be seen in Figure 3a, in the case of allele 1 / allele 2 Hetero amplification (492 bp) generated by the primer annealed in the outer region (outer region) and amplification generated specifically for allele 1 (321) bp), allele 2 specific amplification (232 bp) was observed (lane 1, 2, 3). For the allele 1 homo type, only amplifications (492 bp) generated by primers annealed to the outer region and amplifications (321 bp) specifically generated for allele 1 were observed (lane 4, 5, 6). For the allele 2 homo type, only amplifications (492 bp) generated by primers annealed to the outer region and amplifications specifically generated to allele 2 (232 bp) were observed (lane 7, 8, 9). The 321 bp and 232 bp amplification products were accurately detected by the single nucleotide sequence allele 1 and allele 2 primers designed in this experiment, and no false positive or false negative results were observed. However, according to the conventional method (lower panel of Fig. 3a), a wrong amplification result was obtained.

According to the results of the present invention, according to the method of the present invention, even under the conditions of multiplex PCR, it is possible to accurately distinguish the single nucleotide sequence polymorphism of human genes without false-positive and false-negative errors. Able to know.

According to the method of the present invention, two or more nucleotide variations can be accurately and simultaneously detected without errors through simple amplification reactions without conventional additional restriction enzyme processing or sequencing.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Seegene, Inc. <120> Method for Detecting Nucleotide Variations <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLT-F <400> 1 ctcgtggtgg acttctctca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLT-R <400> 2 gtgtaaaagg ggcagcaaag 20 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> YVDD-R <220> <221> misc_feature (222) (18) .. (22) <223> n denotes deoxyinosine <400> 3 cttggccccc aataccannn nntccacata 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> YIDD-F <220> <221> misc_feature (222) (18) .. (22) <223> n denotes deoxyinosine <400> 4 ccccactgtt tggctttnnn nnatattgat 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> YSDD-F <220> <221> misc_feature (222) (18) .. (22) <223> n denotes deoxyinosine <400> 5 ccccactgtt tggctttnnn nnatagtga 29 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> L528M-R <220> <221> misc_feature (222) (19) .. (23) <223> n denotes deoxyinosine <400> 6 aacaaatggc gctagtaann nnngccatga g 31 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> IC-F <220> <221> misc_feature (222) (21) .. (25) <223> n denotes deoxyinosine <400> 7 taaatcacag gccgaaaccg nnnnnattaa ggggc 35 <210> 8 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> IC-R <220> <221> misc_feature (222) (25) .. (29) <223> n denotes deoxyinosine <400> 8 gtgaatgtga agaagtaggc cgttnnnnng gcaataatg 39 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> CYP-TSP-F <220> <221> misc_feature (222) (25) .. (29) <223> n denotes deoxyinosine <400> 9 agagaagaat tgttgtaaaa agtannnnna ttaatataa 39 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CYP-TSP-R <220> <221> misc_feature (222) (24) .. (28) <223> n denotes deoxyinosine <400> 10 aaactagtca atgaatcaca aatnnnnnag cagtcac 37 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> NVS-Allele2-F <220> <221> misc_feature (222) (23) .. (27) <223> n denotes deoxyinosine <400> 11 taattttccc actatcattg annnnntccc agg 33 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> NVS-Allele1-R <220> <221> misc_feature (222) (22) .. (26) <223> n denotes deoxyinosine <400> 12 caaggttttt aagtaatttg ttnnnnnttc ccggg 35  

Claims (23)

A method of simultaneously detecting at least two or more nucleotide variations of a target nucleotide sequence in a sample comprising a target nucleotide sequence exhibiting a nucleotide variation comprising the following steps: (a) contacting the primers of the following (i) to (iv) with the target nucleotide sequence under hybridization conditions:        (i) a first nucleotide variant specific comprising a nucleotide that specifically hybridizes to a mutation-generating site of a target nucleotide sequence having a nucleotide corresponding to a first nucleotide variant and is complementary to the nucleotide corresponding to the first nucleotide variant Nucleotide variation specific primer 1: NVS-P1;        (Ii) target specific primer 1: TSP1 that hybridizes to a specific site of a target nucleotide sequence located upstream of the mutation-occurring site where said NVS-P1 primer hybridizes;        (Iii) specifically hybridizes to a sequence complementary to a mutation-occurring site of a target nucleotide sequence having a nucleotide corresponding to a second nucleotide variant at the same site, adjacent site or spaced apart site where the first nucleotide variant appears; A second nucleotide variant specific primer (NVS-P2) comprising a nucleotide corresponding to the second nucleotide variant; And         (Iii) a target sequence specific primer 2: TSP2 that hybridizes to a specific site of a target nucleotide sequence located downstream of the mutation-producing site to which the NVS-P2 primer hybridizes, wherein (i) And the primer sets of (ii) and (iii) and (iii) primer sets are selected and designed to produce amplification products of different sizes, and the NVS-P1 and NVS-P2 primers are represented by the following general formula (I) Is displayed: 5'-A _p -Y _q -V _r -3 '(I) In the general formula, A _p is a variation adjacent specificity portion having a hybridization sequence substantially complementary to the target sequence to be hybridized, and Y _q is a separation portion including at least three universal bases. V _r is a variation specificity portion comprising a nucleotide variant corresponding to or complementary to a nucleotide variant and having a hybridization sequence substantially complementary to the target sequence, p, q and r represent the number of nucleotides, X, Y and Z are deoxyribonucleotides or ribonucleotides, the Tm of the variant adjacent specificity zone is higher than the Tm of the variant specificity zone, the partitioning zone has the lowest Tm of the three zones, and the partitioning zone in terms of hybridization specificity Variation specificity zone Variation specificity zone Such specificity cleavage allows the hybridization specificity of the oligonucleotide overall structure to be determined by the variant adjacent specificity region and the variant specificity region, which in turn improves the hybridization specificity of the oligonucleotide overall structure; (b) performing at least two cycles of primer annealing, primer extension and denaturation to amplify the target nucleotide sequence; And, (c) detecting nucleotide variations by confirming the size of the amplification products generated in step (b). The method of claim 1, wherein the universal base is deoxyinosine, inosine, 7-diaza-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, 1- (2'-deoxy-beta-D-ribofura Nosyl) -3-nitropyrrole, deoxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4 -Nitrobenzimidazole, dioxy 4-aminobenzimidazole, 4-aminobenzimidazole, dioxy nebulin, 2'-F nebulin, 2'-F 4-nitrobenzimidazole, PNA-5 Introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebulinine, morpho Lino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, Phosphoramidate-nebulin, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethylinosine, 2 '-0-methoxyethyl nebulin, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro-benzimidazole, 2'-0-methoxyethyl 3- Nitropyrrole and a combination of said bases. The method of claim 2, wherein the universal base is deoxyinosine, inosine, 1- (2'-deoxy-beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole. . The method of claim 1, wherein said variant adjacent specificity region is 15-40 nucleotides in length. The method of claim 1, wherein the variant specificity region is 3-15 nucleotides in length. The method of claim 1, wherein the partitioning zone has a length of 3-10 nucleotides. The method of claim 1, wherein the variant adjacent specificity zone has a Tm of 40-80 ° C. 3. The method of claim 1, wherein the variant specificity zone has a Tm of 10-40 ° C. 3. The method of claim 1, wherein the partition zone has a Tm of 3-15 ° C. The method of claim 1, wherein the nucleotide corresponding to or complementary to the nucleotide variant is located at the 3′-end of the variant specificity region or 1 to 10 bases away from the 3′-end. The method of claim 10, wherein the nucleotide corresponding to or complementary to the nucleotide variant is located 2 to 7 bases apart from the 3′-end of the variant specificity region. 12. The method of claim 11, wherein the nucleotide corresponding to or complementary to the nucleotide variant is located 3 to 6 bases apart from the 3'-end of the variant specificity region. The method of claim 10, wherein the nucleotide corresponding to or complementary to the nucleotide variation is located in the central portion of the variation specificity region. The method of claim 1, wherein the target sequence specific primer (TSP) has a structure represented by Formula I wherein the variant specificity region is substantially complementary to the target sequence without corresponding or complementary nucleotide variations. Characterized by having a hybridization sequence. The method of claim 1, wherein the variant specificity regions of the NVS-P1 primer and the NVS-P2 primer comprise sequences that partially overlap each other. The method of claim 1, wherein step (a) additionally uses one or more nucleotide variation specific primers that hybridize to other mutations other than those in which the NVS-P1 and NVS-P2 primers hybridize. The method characterized in that carried out. The method of claim 1, wherein step (a) is performed by additionally using a pair of primers to generate an internal control by amplifying a nucleotide sequence other than the target nucleotide sequence. The method of claim 1, wherein the method is a method for detecting drug resistant pathogens. 19. The method of claim 18, wherein the pathogen is human immunodeficiency virus (HIV) -1, HIV-2, hepatitis B virus (HBV), hepatitis C virus (HCV) or human herpesvirus (HVV). 19. The method of claim 18, wherein the medicament is zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delavirdine, Efavirenz, adefovir, adefovir dipivoxil, FTC, D4FC, BCH-189, F-ddA, tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -one (tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -one), tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -thione), (S) -4-isopropoxycarbo Neyl-6-methoxy-3- (methylthiomethyl) -3,4-dihydroquinoxaline-2 (1H) -thione ((S) -4-isopropoxycarbonyl-6-methoxy-3- (methylthiomethyl) -3 , 4,-dihydroquinoxaline-2 (1H) -thione), saquinavir, ritonavir, indinavir, nelfinavir amprenavir, ente Selected from the group consisting of entecavir, famciclovir, benzo-1,2,4-thiadiazine antiviral agent, ribavirin and interferon At least one antiviral agent. The method of claim 20, wherein the method is for simultaneously detecting at least two or more nucleotide variations that cause lamivudine resistance of HBV. 22. The method of claim 21, wherein the nucleotide variation that induces lamivudine resistance is a mutation at the 552th codon of the polymerase gene of HBV. 23. The method of claim 22, wherein the NVS-P1 primer or NVS-P2 primer is selected from SEQ ID NOs: 3-6.

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