KR100977186B1 - Processes Using Dual Specificity Oligonucleotide and Dual Specificity Oligonucleotide - Google Patents

Abstract

The present invention provides a variety of methods and three different T _m by template-dependent extension reactions using bispecific oligonucleotides. It relates to a bispecific oligonucleotide comprising a site. According to the present invention, features of bispecific oligonucleotides with high hybridization specificity and mismatch tolerance are highlighted.

Bispecificity, Oligonucleotides, Annealing, Microarrays

Description

Processes using dual specificity oligonucleotide and dual specificity oligonucleotide

The present invention relates to various methods and dual specific oligonucleotides using dual specific oligonucleotides. More specifically, the present invention relates to various methods and bispecific oligonucleotides by template-dependent extension reactions using bispecific oligonucleotides.

Nucleic acid amplification is an essential process in various methods used in the field of molecular biology, and various amplification methods have been suggested. For example, Miller, HI et al. (WO 89/06700) amplify nucleic acid sequences comprising hybridizing a promoter / primer sequence to target single-stranded DNA (“ssDNA”) and then transcribing many RNA copies of that sequence. A method is disclosed. Other known nucleic acid amplification methods include transcriptional amplification systems (Kwoh, D. et al., Proc . Natl . Acad . Sci . USA , 86: 1173 (1989); and Gingeras TR et al., WO 88/10315 ).

The most widely used nucleic acid amplification method known as polymerase chain reaction (hereinafter referred to as "PCR") is the repetition of denaturation of double stranded DNA, annealing oligonucleotide primers into DNA templates and primer extension by DNA polymerase. Cycle procedures (Mullis et al., US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354.) Oligonucleotide primers used for PCR are DNA templates. It is designed to anneal to the opposite strand of: The primer is extended by DNA polymerase and the extension product acts as a template strand for other primers in the following process: PCR amplification results in an exponential increase in DNA fragments and amplification The length of the DNA fragment thus obtained is determined by the 5'-end of the oligonucleotide primer.

Since success in nucleic acid amplification, particularly PCR amplification, depends on the specificity of one primer to anneal only to its target sequence, it is important to optimize such intermolecular interactions. Whether a primer anneals only to its complete complement or also to sequences having one or more mismatch nucleotide sequences can be determined depending on the annealing temperature. In general, the higher the annealing temperature, the higher the specific annealing probability of the primer for the complete match template, and thus the higher the chance that only the target sequence will be amplified. Conversely, there is a possibility that more mismatches between the template and the primer occur at low annealing temperatures. In view of this phenomenon, adjusting the annealing temperature can change the binding specificity between the template and the primer. For example, if there is no product, the temperature may be too high to anneal. If products of various lengths occur in a control in which only one primer is present, these results indicate that the single primer anneals to one or more sites of the template. In this case, it is preferable to raise the annealing temperature.

 In addition to the annealing temperature, primer search parameters such as primer length, GC amount and length of PCR product should be considered for the annealing specificity of the primer. If primers satisfying the above mentioned parameters are used, primer annealing is specified, the annealing specificity of the primer in target DNA amplification is significantly improved, and the problems of background and non-specific products caused by the primer are solved. Well-designed primers not only solve non-specific annealing and background problems, but also allow to distinguish cDNAs or genomic templates from RNA-PCR.

Many methods have been developed to improve primer annealing specificity and to allow for amplification of the desired product. For example, a touch-down PCR (Don et al., ( 1991) Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res., 19, 4008), hot start PCR (DAquila et al., ( 1991) Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucleic Acids Res., 19, 3749), neseuti agent PCR (Mullis and Faloona, (1987 ) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol . 155, 335-350) and booster PCR (Ruano et al., (1989) Biphasic amplification of very dilute DNA samples via booster PCR. Nucleic Acids Res . 17, 5407).

Other approaches have also been reported, which use various enhancer compounds to improve the specificity of PCR. Enhancer compounds include chemicals that increase the effective annealing reaction temperature, DNA binding proteins, commercially available reactants, and the like. However, there are no magical additives that can guarantee success in any PCR, and testing other additives under various conditions, such as annealing temperatures, is a tedious task. Although the approaches described above contribute to some degree in improving primer annealing specificity, the methods described above provide a fundamental solution to the problems resulting from primers used for PCR amplification, such as non-specific products and high background. I can't.

PCR-related techniques are widely used in a variety of applications and methods in biological and medical research, as well as amplification of target DNA sequences, such as reverse transcriptase PCR (RT-PCR), differential display PCR (DD-). PCR), cloning of known or unknown genes, rapid amplification of cDNA ends (RACE), arbitrary priming PCR (AP-PCR), multiplex PCR, SNP genomic typing, PCR-associated genome Assay (McPherson and Moller, (2000) PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY).

Although improved approaches have been continuously introduced for each method, all methods and techniques, including nucleic acid amplification, in particular PCR amplification processes, as described above, have limitations and problems resulting from the non-specificity of the primers used, such as It is not completely free from false positives, deterioration of reproducibility and high background problems.

DNA hybridization, on the other hand, is a fundamental process in molecular biology and is affected by ionic strength, base composition, length of fragments to which nucleic acid molecules bind, degree of mismatch and presence of denaturing agents. This technique using DNA hybridization is very useful for determining specific nucleic acid sequences and is of great value in clinical diagnosis, genetic research, and forensic analysis. For example, Wallance and co-workers can fully distinguish subtle sequence differences, such as single base changes, using short oligomers (eg, 14-mers), which can be used for molecular analysis of point mutations in β-globin genes. (Wallace, BR, et al., (1981) The use of synthetic oligonucleotides as hybridization probes.Hybridization of oligonucleotides of mixed sequence to rabbit β-globin DNA.Nucleic Acids Res . 9, 879-894; And Conner, BJ, et al. (1983) Detection of sickle cell β-globin allele by hybridization with synthetic oligonucleotides. Proc . Natl . Acad . Sci . USA 80, 278-282).

Despite the powerful power of oligonucleotide hybridization methods to accurately identify complementary strands, this technique still faces limitations. Hybrids containing oligonucleotides are much more unstable than hybrids of long nucleic acids, which are reflected at low melting temperatures. The instability of the hybrid is one of the most important factors to consider when designing oligonucleotide hybridization. The difference in stability between the perfectly matched complement and the complement that only mismatches one base is so small that about 0.5 ° C. difference occurs in T _m s (dimer melting temperature). The shorter the oligomer, the shorter (which enables the identification of complementary strands in more complex mixtures), the greater the effect of single base mismatch on the stability of the overall dimer. However, a disadvantage of these short oligonucleotides is that they hybridize weakly to fully complementary sequences. Thus, in view of this, hybridization should be carried out under reduced stringency conditions, which in turn results in a greatly reduced specificity of hybridization.

Many efforts have been made to improve the specificity of oligonucleotide hybridization. Improve the ability to chemically denature DNA bases for high-sensitive hybridization (Azhikina et al., Proc . Natl . Acad . Sci ., USA , 90: 11460-11462 (1993)) and the ability to distinguish mismatches Hybridization is performed at a low temperature for a long time and then washed (Drmanac et al., DNA and Cell Biology , 9: 527-534 (1990)). Recently, other methods have been introduced that can increase the degree of differentiation of mononucleotide polymorphisms (SNPs) in DNA hybridization using artificial mismatches (Guo et al., Nature Biotechnology , 15: 331-5 (1997)). In addition, a number of US patents, including US Pat. Nos. 6,077,668, 6,329,144, 6,140,054, 6,350,580, 6,309,824, 6,342,355 and 6,268,128, disclose probes for hybridization and their applications. For each method developed, improved approaches are constantly being introduced, but not all methods and techniques, including oligonucleotide hybridization, can be completely free from limitations and problems with nonspecific oligonucleotide hybridization.

Moreover, artificial factors such as spotting and immobilizing oligonucleotides on the substrate and failure to establish optimal hybridization conditions are likely to affect the negative data of hybridization. In particular, the effect of false results is large in the results obtained with the high throughput screening method. Artificial factors inherent in spotting and hybridization are major drawbacks of oligonucleotide-based DNA microarrays.

In addition, the development of DNA sequencing techniques with improved speed, sensitivity and speed is of great importance in biological research. Traditional DNA sequencing methods, developed more than 20 years ago (Sanger et al. Proc . Natl . Acad . Sci . , 74: 5463-5467), have limited speed and cost in additional applications. Therefore, several new technologies have been proposed. Three promising methods include sequencing by hybridization (Brain and Smith, J. Theor. Biol ., 135: 303-307 (1988); Dramanac et al., Genomics , 4: 114-128 (1989); Sourthern, EM Patent WO / 10977 (1989)), parallel signature sequencing based on ligation and cleavage (Brenner et al., Proc. Natl . Acad Sci . , 97: 1665-1670 (2000)) and pyrosequencing (Ronaghi et al., Anal . Biochem . , 242: 84-89 (1996); Science 281: 363-365 (1998)). . The success of sequencing in this technique is absolutely dependent on the hybridization specificity of the sequencing primer to the target nucleic acid. Given the hybridization specificity of the sequencing primers, current methods have limitations in the length of template nucleic acid that is put in to perform the sequencing reaction. Generally, in order to extend after specific hybridization of sequencing primers, the sequencing reaction is preferably performed using nucleic acid templates smaller than several hundred base pairs.

However, for advanced research, increased speed, sensitivity, and large-scale DNA sequencing should not be limited to the size of the nucleic acid template. In this regard, if the sequencing primer is hybridized with the target nucleic acid with high specificity, direct sequencing of one target nucleic acid from the population of nucleic acid templates is possible.

Throughout this specification many patent documents and articles are referenced and their citations are indicated. The disclosures of cited patent documents and articles 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.

In order to eliminate the problems and disadvantages of conventional oligonucleotides used as primers or probes, and various methods involving nucleic acid hybridization processes, we have invented bispecific oligonucleotides that perform template-dependent reactions with higher specificity. Its excellent applicability has been demonstrated in a variety of methods including oligonucleotide hybridization or annealing.

It is therefore an object of the present invention to provide a method for preparing nucleic acid molecules by template-dependent extension reactions using bispecific oligonucleotides.

Another object of the present invention is to provide a method for selectively amplifying a target nucleic acid sequence in one DNA or nucleic acid mixture.

Another object of the present invention is to provide a method for amplifying two or more target nucleotide sequences simultaneously using two or more primer pairs in the same reaction.

Another object of the present invention is to provide a method for sequencing nucleic acid molecules in one DNA or nucleic acid mixture.

It is still another object of the present invention to provide a method for detecting a nucleic acid molecule having genetic diversity by a template-dependent extension reaction.

Another object of the present invention is to provide a method for detecting a target nucleotide sequence in a nucleic acid sample using a bispecific oligonucleotide-fixed microarray.

Another object of the present invention is to provide a bispecific oligonucleotide for preparing a nucleic acid molecule by a template-dependent extension reaction.

Another object of the present invention is to provide a method by which the annealing specificity of the oligonucleotide can be determined in duplicate through the oligonucleotide structure.

It is yet another object of the present invention to provide a method for improving the annealing specificity of oligonucleotides.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The present invention relates to (a) various methods using bispecific oligonucleotides and (b) bispecific oligonucleotides used in the methods. The bispecific oligonucleotide of the present invention (hereinafter referred to as “DS oligo”) allows the primer or probe to be annealed to the target nucleic acid with improved specificity, thereby significantly improving the specificity and hybridization reaction of nucleic acid amplification (particularly PCR). do.

Bispecific oligonucleotides ( DS Oligo )

According to one aspect of the invention, the invention provides a bispecific oligonucleotide for synthesizing a nucleic acid molecule by a template-dependent extension reaction, represented by the following general formula:

5'-X _p -Y _q -Z _r -3 '

X _p represents a 5′-high T _m specificity site having a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid being hybridized, Y _q represents a cleavage site comprising at least two universal bases, and Z _r Denotes a 3′-low T _m specificity site having a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid to hybridize, p, q and r are the number of nucleotides, and X, Y and Z are deoxyribonucleotides or Ribonucleotides; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a non-base pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the template nucleic acid, and thus, in terms of annealing specificity for the template nucleic acid. '- high T _m specificity portion and the 3 ' to be separate from the low T _m specificity portion, the annealing specificity of the oligonucleotide is 5'-high T _m specificity determined by both the site and the 3'-low T _m specificity portion This increases the overall annealing specificity of the oligonucleotides.

As used to refer to DS oligonucleotides (hereinafter referred to as "DS oligo"), the term "dual specificity" is an acronym for expressing the characteristics of DS oligonucleotides, the annealing specificity of DS oligonucleotides to a target sequence. , It is determined in duplicate by two separate sites, namely the 5'-high T _m specific site and the 3'-low T _m specific site. In general, the annealing specificity of a primer or probe is governed by its entire sequence. In contrast, the annealing specificity of DS oligo is determined in duplicate by two sites separated by cleavage sites (5'-high T _m specific sites and 3'-low T _m specific sites), wherein these three sites are one oligonucleotide. Located in the sequence. This dual specificity allows DS oligos to exhibit higher specificity as primers and probes, which makes the invention novel and inventive with respect to the prior art.

On the other hand, the inventor has already developed an annealing control primer (ACP) to increase the annealing specificity as disclosed in WO 03/050303, the teachings of which are incorporated herein by reference. The DS oligo of the present invention is clearly different from ACP in the following aspects: (iii) DS oligo has two specificity sites to hybridize with the target sequence while ACP has one specificity site; (Ii) three sites in DS oligo are T _m While clearly distinguished in terms, the sites in the ACP are not; (Iii) DS primers are extended to synthesize nucleic acid molecules complementary to the template only when annealing occurs at both the 5'-high T _m specific site and the 3'-low T _m specific site, whereas ACP is extended to the 3'-terminal site. Even when annealing occurs; (Iii) Thus, the extension or hybridization specificity of DS oligo is determined in duplicate by two split sites, namely the 5'-high T _m specific site and the 3'-low T _m specific site, whereas in the case of ACP only 3 ' It is controlled by only the terminal part. Thus, the annealing or hybridization specificity for the target sequence of DS oligo is much greater than APC, indicating that DS oligo is novel and progressive for ACP.

The salient feature of DS oligo is that it has three different sites with distinctive features within one oligonucleotide molecule: 5'-high T _m specific site, 3'-low T _m specific site and cleavage site.

DS oligos are useful for a variety of methods and assays, including template-dependent prolongation. As used herein, the term “template-dependent extension reaction” means a reaction that extends an oligonucleotide molecule hybridized to a target sequence, which is accomplished by binding consecutive nucleotides to the ends of the oligonucleotides. In this case, the extended sequence is determined by the complementary template sequence.

Representative examples of principles illustrating hybridization (annealing) specificity of DS oligos are shown in FIG. 1A. Referring to Figure 1A, DS oligo will be described in more detail.

If only the 5'-high T _m specific site of DS oligo is annealed to the template, the site cannot serve as a priming site for template-dependent extension and eventually no extension reaction occurs.

The 5'-high T _m specific site of the DS oligo is annealed to the non-target sequence, while the 3'-low T _m specific site with shorter sequences does not anneal to the non-target sequence. This is because the 5'-high T _m specific site and the 3'-low T _m specific site are separated by cleavage sites in terms of annealing. That is, the 3'-low T _m specific site is involved in annealing in a manner that is relatively independent of the 5'-high T _m specific site, and the annealing of the 3'-low T _m specific site is 5'-high T _m specificity. Less affected by annealing of the site. In this context, the likelihood that the 3′-low T _m specificity site will anneal to the non-target sequence is very low.

If only the 3'-low T _m specific site has a sequence complementary to the non-target site, no annealing will occur under certain high stringency conditions such as stringent conditions for annealing 5'-high T _m specific sites. According to a preferred embodiment, it is advantageous to carry out the template-dependent extension using DS oligo under stringent conditions with annealing temperatures higher than the T _m of the 3'-low T _m specific site.

If both the 5'-high T _m specific site and the 3'-low T _m specific site have sequences that are substantially complementary to the template, the DS oligo can be annealed to the template and thus a successful extension reaction occurs.

Without being bound by theory, it is believed that the cleavage site makes the 3′-low T _m specific site more sensitive to annealing conditions (eg, temperature and sequence complementarity). Thus, the likelihood of non-specific hybridization between the 3′-low T _m specific site and the non-target sequence is even lower under certain annealing (or stringent) conditions. If the 5'-high T _m specific site as well as the 3'-low T _m specific site are annealed to its target sequence, the 3'-terminus of the 3'-low T _m specific site is positioned to be extended by DNA polymerase. Make.

As used herein, the term “oligonucleotide” refers to a linear oligomer of natural or modified monomers or linkages, and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to target nucleotide sequences. It is either naturally present or artificially synthesized. Oligonucleotides are preferably single chain for maximum efficiency in hybridization. Preferably, the oligonucleotide is an oligodioxyribonucleotide. Oligonucleotides of the invention may include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. Oligonucleotides may also include ribonucleotides. For example, 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'-0-alkyl DNA, 2'-0-allyl DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohex Tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, Thityl-, propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents being fluoro-, bromo-, chloro- , EO also -, methyl-, ethyl-, vinyl-, formyl-, alkynyl -, alkenyl-, thiazol quinolyl and quinoxalyl-, already quinolyl and quinoxalyl -, pyridyl-), may comprise inosine and diamino purine.

As used herein, the term “primer” refers to an oligonucleotide, which is a condition in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, i.e., the presence of a polymerizer such as nucleotide and DNA polymerase, and It can serve as a starting point for the synthesis at 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.

The primer should be long enough to prime the synthesis of the extension product in the presence of the polymerizer. Suitable lengths of the primers depend on a number of factors, such as temperature, application and source of the primer. The term “annealing” or “priming” refers to the placement of an oligodioxynucleotide or nucleic acid in a template nucleic acid, where the polymerase polymerizes the nucleotide to form a nucleic acid molecule that is complementary to the template nucleic acid or portion thereof. Let's do it. As used herein, the term “hybridization” means the formation of double-stranded nucleic acids from complementary single-stranded nucleic acids. The terms “annealing” and “hybridization” do not differ and are used interchangeably herein.

As used herein, the term “probe” is a single chain nucleic acid molecule that includes a site substantially in a target nucleotide sequence.

The term "portion" as used herein referring to the DS oligo of the present invention means a nucleotide sequence separated by a cleavage site. The term "5'-high T _m specific site (5'-high T _m) specificity portion) "or the term"3'-low T _m specificity portion _{(3'-low T m specificity portion} ) " refers to a nucleotide sequence in each of the 5'-end and 3'-end of the DS oligo of this invention, and The term 5'-high T _m specific site ", which is used to refer to DS oligos, has the highest T _m of the three sites and is substantially complementary to the position of the template nucleic acid hybridization nucleotides. Means a site having a sequence. With respect to the DS oligo than "3'-low T _m specificity portion _{(3'-low T m specificity portion} )" is has the lowest T _m than the 5'-high T _m specificity portion, cleavage site with a high T _m, By a site having a hybridizing nucleotide sequence substantially complementary to one position of the template nucleic acid.

As used herein, the term “T _m ” means a melting temperature at which half of the nucleic acid double-stranded molecules become single stranded. The term, referring to parts of the DS oligo "high T _m (T _m high)" and "low T _m (low T _m)" is absolute T _m It means the relative T _m value, not the value. That is, the 5'-high T _m specificity portion of the 3'-low T _m T _m only be just higher than the relative demand of the region T _m specificity.

The 5′-high T _m specificity site and the 3′-low T _m specificity site are designed to have a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid to hybridize. With respect to DS oligo, the term “substantially complementary” means that the oligonucleotides are sufficiently complementary to selectively hybridize to the template nucleic acid sequence under specified annealing conditions or stringent conditions, such that the annealed oligonucleotides are incorporated into the polymerase. Means that it can be extended to form a copy complementary to the mold. Thus, this term has a different meaning from the term “completely complementary” or related thereto. The 5'-high T _m specificity site and the 3'-low T _m specificity site of the DS oligo may have one or more mismatches with respect to the template, within the range in which the DS oligo can act as a primer or prab. Most preferably, the 5'-high T _m specific site and / or 3'-low T _m specific site of the DS oligo have a nucleotide sequence that is completely complementary to one position of the template, i.e., no mismatch.

For the successful implementation of DS oligo it is essential that the T _m of the 5'-high T _m specific site be higher than the T _m of the 3'-low T _m specific site. Preferably, the 5'-high T _m specificity portion of the T _m is 40-80 ℃, more preferably, 40-75 ℃, and more preferably 50-68 ℃, and most preferably from 50-65 ℃ . Preferably, the 3'-low T _m specificity portion T _m is 10-40 ℃, more preferably, 15-40 ℃, and most preferably from 20-35 ℃. Preferably, the 5'-high T _m specificity portion of the 3'-low T _m T _m T _m specificity portion of the More preferably at least 3 ° C., more preferably at least 10 ° C., even more preferably at least 15 ° C., and most preferably at least 20 ° C. high. Advantageously, the 5'-high T _m specificity portion of the T _m of the 3'-low T _m T _m specificity portion Higher 5-70 ° C, preferably 10-70 ° C, more preferably 10-60 ° C, even more preferably 10-50 ° C, even more preferably 10-40 ° C and most preferably 20 -40 ℃ high.

According to a preferred embodiment, the 5'-high T _m specificity site is longer than the 3'-low T _m specificity site. The 5′-high T _m specificity site preferably has 15 to 40 nucleotides in length, more preferably 15 to 30 nucleotides, most preferably 20 to 25 nucleotides in length. The 3′-low T _m specificity site preferably has a length of 3 to 15 nucleotides, more preferably 5 to 15 nucleotides, and most preferably 6 to 12 nucleotides in length.

The cleavage site comprising at least two universal bases is a factor in determining the benefits and characteristics of DS oligos. As used herein, the term "universal base" refers to a base capable of forming base pairs with each of the natural DNA / RNA bases without distinction to the natural DNA / RNA bases.

It is known that nucleotides of uncertain positions in degenerate primers are replaced by universal bases, which are known as deoxyinosine (Ohtsuka et al, (1985) J. Biol . Chem . 260, 2605-2608; Sakanari et. al., (1989) Proc . Natl. Acad . Sci . 86, 4863-4867, 1- (2'-deoxy-betaD-ribofuranosyl) -3-nitropyrrole (Nichols et al., ( 1994) Nature 369, 492-493) and 5-nitroindole (Loakes and Brown, (1994) Nucleic Acids Res . 22, 4039-4043, which bases are nonspecifically base paired with four conventional bases and are used to solve problems associated with the design of degenerate primers. However, these universal bases form one site in the oligonucleotide molecule to create a bubble structure during annealing (hybridization) or amplification and then separate two opposite adjacent sequences, resulting in bispecificity through two split specificity (annealing) moieties. There is no research that can increase the annealing specificity of the primer or probe to the target sequence.

According to a preferred embodiment, the universal base of the cleavage site is 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, 1- ( 2'-deoxy-beta-D-ribofuranosyl) -3-nitropyrrole, deoxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitro Indole, dioxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, dioxy 4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2'-F nebula Rin, 2'-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino -5-nitroindole, morpholino-nebulin, morpholino-inosine, morpholino-4-nitrobenzimidazole, Leporino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebulin, phosphoramidate-inosine, phosphoramidate-4-nitro Benzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethyl inosine, 2'-0-methoxyethyl nebulin, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro-benzimidazole, 2'-0-methoxyethyl 3-nitropyrrole and combinations of these bases. More preferably said universal base or non- discriminatory base analog is deoxyinosine, 1- (2'-deoxy-beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole, most Preferably dioxyinosine.

The universal base at the cleavage site can be included in a continuous manner or can be included intermittently with other nucleotides such as dNMPs. Preferably, said cleavage site comprises a sequential sequence of nucleotides having a universal base, preferably deoxyinosine.

It is essential that the cleavage site in DS oligo has the lowest T _m of the three sites, whereby the cleavage site is annealed under conditions where the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the template nucleic acid. It forms a non-base pair bubble structure and in terms of annealing specificity for template nucleic acids, the 5'-high T _m specificity site is separated from the 3'-low T _m specificity site, and the annealing specificity of the oligonucleotide is 5'-high T Double determination by the _m specificity site and the 3′-low T _m specificity site significantly improves the overall annealing specificity of the oligonucleotide. Preferably the T _m of the cleavage site is 3-15 ° C, more preferably 4-15 ° C, and most preferably 5-10 ° C.

According to a preferred embodiment, the cleavage site between the 5'-high T _m specific site and the 3'-low T _m specific site comprises at least three universal bases, more preferably at least four universal bases, and most Preferably at least 5 universal bases. According to a preferred embodiment, the cleavage site comprises 2-10 universal bases, more preferably 3-10 universal bases, even more preferably 4-8 universal bases, and most preferably 5 -7 universal bases.

When primers or probes with longer sequences are needed, the benefits of DS oligo are most pronounced. For example, according to the prior art, primers having a nucleotide sequence of 35 bp or more as the hybridization sequence are likely to generate non-specific amplicons. In contrast, DS oligos produce only specific amplifications, even when they have long sequences, because DS oligos have two hybridization sequences (i.e., 5'-) each separated in terms of their interaction with the template (i.e. annealing). High T _m specificity site and 3′-low T _m specificity site). For example, the DS oligo may comprise a 35-45 bp hybridization sequence that is complementary to the target sequence. In view of this, it can be seen that the present invention enables the design of primers with longer sequences that are considered impractical in conventional primer design strategies.

According to a preferred embodiment, the 5'-high T _m specificity site is 15 to 25 nucleotides in length, the cleavage site is 3 to 10 nucleotides in length and the 3'-low T _m specificity site is 3 to 15 nucleotides in length.

More preferably, the 5′-high T _m specificity site is 15 to 25 nucleotides in length, the cleavage site is 3 to 10 nucleotides in length and the 3′-low T _m specificity site is 5 to 15 nucleotides in length. Most preferably, the 5′-high T _m specificity site is 15 to 25 nucleotides in length, and the cleavage site is 5 to 7 nucleotides in length; And the 3′-low T _m specificity site is 6 to 10 nucleotides in length. According to the typical and exemplary DS oligos described in the examples, the 5'-high T _m specificity site is about 20 nucleotides in length; The cleavage site is about 5 nucleotides in length; The 3′-low T _m specificity site is about 8-10 nucleotides in length.

In the most preferred embodiment, DS oligo is represented by the general formula: 5'-X _p- (dI) _q -Z _r -3 '(X _p And the definitions and characteristics of Z _r are as described above, dI denotes deoxyinosine, (dI) _q denotes a cleavage site comprising a sequence of nucleotides having a universal base and q is an integer of 5-7). .

Interestingly, the DS oligos of the present invention have mismatch tolerance under stringent conditions that tolerate mismatches to the target sequence.

Structural representatives of the principles governing mismatch tolerance of DS oligos are shown in FIG. 1B. The 5'-high T _m specificity portion and 3'-low T _m under the specific region is that for both annealing to the template, the 5'-high T _m specificity portion has the one or more, preferably one to three base mismatches Is allowed. Under the 3'-low T _m specificity portion has one or more, preferably one or two base mismatch, the 5'-high T _m specificity portion and 3'-low T _m specificity portion are annealed to the template for both the May be acceptable. Furthermore, at least one, preferably at 5'-high T _m specific site and 3'-low T _m specific site, under conditions in which both the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the template. One to five base mismatches can be tolerated.

In order to impose mismatch tolerance on the DS oligo, the annealing conditions, in particular the annealing temperature, are important. Annealing is performed under conditions where no annealing occurs by the 3′-low T _m specific site alone, and at least one 5′-high T _m specific site and / or 3′-low T _m specific site relative to the target position. With (but limited number) mismatched bases, annealing by all sites occurs. DS oligos with mismatch tolerance are required to amplify or detect nucleotide sequences with genetic variability. DS oligos with mismatch tolerance can be annealed to target sequences that exhibit genetic variability, resulting in successful amplification and detection of the desired nucleotides. That is, DS oligos, originally developed to dramatically increase the specificity of annealing and hybridization, can also be used in methods that require mismatch tolerance under conditions where annealing or stringent conditions are suitably controlled.

According to another aspect of the present invention, there is provided a method of allowing the annealing specificity of an oligonucleotide to be determined dually by the structure of an oligonucleotide comprising the steps of: (a) selecting a target nucleic acid sequence; (b) design a sequence of oligonucleotides having a hybridization sequence substantially complementary to the target nucleic acid and (ii) a cleavage site comprising at least two universal bases such that the cleavage site is sandwiched between the hybridization sequences ( intervene) forming three sites in the oligonucleotide; And (c) the 5′-direction portion of the cleavage site has a higher T _m than the 3′-direction region of the cleavage site, and the cleavage site has the lowest T _m at the three sites. Determining the location of said cleavage site in which (i) the 5'-high T _m specific site of said oligonucleotide has a hybridizing nucleotide sequence substantially complementary to said target nucleic acid, and (ii) said oligonucleotide The 3′-low T _m specificity site has a hybridizing nucleotide sequence substantially complementary to the target nucleic acid; And (iii) the cleavage site between the 5′-high T _m specific site and the 3′-low T _m specific site comprises at least two universal bases; And the 5'-high T _m specificity portion of the 3'-low T _m is the T _m specificity portion is higher than the T _m of the import of the lowest T _m among the cleavage site comprises three parts, different from each other T _m value An oligonucleotide having three distinct sites, wherein the annealing specificity for the target nucleic acid of the oligonucleotide by the above configuration is characterized by the 5'-high T _m specific site and the 3'-low T _m specific site Determined double.

The method provides a novel approach to dramatically increase the annealing specificity of oligonucleotides to hybridize with the target sequence. The method may also be expressed as a method for improving the annealing specificity of oligonucleotides. Furthermore, the present invention can be expressed by a method using a cleavage site comprising at least two universal bases to improve the annealing specificity of oligonucleotides that hybridize to the target sequence.

The present invention is used to prepare the DS oligo described above. Thus, in order to avoid unnecessary repetition, common content between them is not repeated, but common content is inserted in the detailed description of the method of the present invention.

Most conventional methods for designing primers or probes use only sequences that can hybridize to the target sequence. In addition, in order to increase the annealing specificity of the oligonucleotides, amplification or hybridization conditions such as temperature and ion concentration were conventionally adjusted.

In contrast, the methods of the present invention provide new strategies for increasing annealing specificity by introducing new features into the oligonucleotides themselves. The term "through a structure of the oligonucleotide" as used herein in connection with which the annealing specificity of the oligonucleotide can be determined in duplicate gives the oligonucleotide a new feature that allows the annealing specificity to be determined in duplicate. This means that the structure of the oligonucleotide greatly contributes to the annealing specificity of the oligonucleotide.

In the method of the present invention, it is important to design in a sequence of oligonucleotides having a hybridization sequence substantially complementary to the target nucleic acid and (ii) a cleavage site comprising at least two universal bases. In this step, the structural outline of the oligonucleotide is represented by the 5'-terminal site / divided site / 3'-terminal site. The 5′-terminal and 3′-terminal two parts have hybridization sequences substantially complementary to the target nucleic acid and the cleavage site intervenes therebetween.

The most important step in the present invention is that the 5'-direction side of the splitting site has a higher T _m than the 3'-direction side of the splitting site, and the splitting site has the lowest T _m at the three sites. Determining the position of the cleavage site in the oligonucleotide, thereby providing an oligonucleotide having three distinct sites with different T _m values.

The new structural features inserted into the oligonucleotides by the method are as follows: (iii) three distinct sites (5'-high T _m specific site, cleavage site and 3'-low T _m specific site) in the oligonucleotide sequence. ); (Ii) three different T _m values; (Iii) a cleavage site between the 5′-high T _m specific site and the 3′-low T _m specific site and comprising at least two universal bases; (Iii) two sites separated on the annealing side by the cleavage site and interact with the target in the annealing step; (Ⅴ) 5'- high T _m specificity portion, 3'-low T _m specificity portion and the cleavage site in order T _m value. This structural feature allows the annealing specificity of the oligonucleotides finally provided by the present invention to be determined dually by two parts, the 5'-high T _m specific site and the 3'-low T _m specific site, which is dependent on the target sequence. The annealing specificity of the oligonucleotides for these is to be greatly increased.

Oligonucleotides designed and prepared according to the methods of the present invention exhibit much higher annealing specificities than do not have the three sites described above.

The features and benefits of DS oligo can be explained as follows:

(a) The cleavage site of the DS oligo contains at least two universal bases, resulting in the lowest T _m in DS oligo, with the 5'-high T _m specific site and the 3'-low T _m specific site templated. And to form a non-base pair bubble structure under conditions annealed to the nucleic acid. Such non base pairing bubble structure is such that the 5'-high T _m specificity portion separated from the 3'-low T _m specificity portion in terms of annealing specificity;

(b) 5'- high T _m specificity portion is higher than the T _m of the 3'-low T _m specificity portion T _m, is the cleavage site having a low T _m, is characterized by the 3'-low T _m specificity portion is Make it possible to set stringent conditions in which annealing by itself does not occur;

(c) Thus, the overall annealing specificity of DS oligo is determined in duplicate by both the 5'-high T _m specific site and the 3'-low T _m specific site;

(d) Eventually, the overall annealing specificity of DS oligo is greatly increased.

The DS oligo of the present invention can be used in various fields, such as Miller, HI method (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR, Wu, DY et al., Genomics 4: 560 (1989)), polymerase ligase chain reaction (Barany, PCR Methods and Applic., 1: 5-16 (1991)), gap-LCR (WO 90/01069), repair chain reaction (EP 439,182) ), Primer-related nucleic acid amplification methods such as 3SR (Kwoh et al., PNAS, USA, 86: 1173 (1989)) and NASBA (US Pat. No. 5,130,238), and (ii) cycle sequencing (Kretz et al. ., (1994) Cycle sequencing . PCR Methods Appl . 3: S107-S112) and pyro sequencing (Ronaghi et al., (1996) Anal . Biochem ., 242: 84-89; and (1998) Science 281: 363-365), etc., and hybridization-related techniques such as (iii) detection of target nucleotide sequences using oligonucleotide microarrays.

The DS oligo of the present invention can be applied to various nucleic acid amplification, sequencing and hybridization-related techniques. Representative examples demonstrating the applicability of DS oligos include:

Ⅰ. Application to Nucleic Acid Molecule Preparation

According to another aspect of the present invention, the present invention provides a method for preparing a nucleic acid molecule by a template-dependent extension reaction using a bispecific oligonucleotide comprising the following steps:

(a) annealing the bispecific oligonucleotide to a template nucleic acid molecule, wherein the bispecific oligonucleotide has a 5'-high T _m specificity site (5'-high T _m). specificity portion), 3'-low T _m specificity portion (5'-low T _m a specificity portion and a separation portion, three sites, wherein the 5'-high T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid to hybridize The site comprises at least two universal bases and the 3′-low T _m specific site has a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the template nucleic acid, and the annealing is a 3'-low T _m specific site. It is carried out under conditions in which annealing by itself does not occur; And

(b) extending said bispecific oligonucleotide to produce a nucleic acid molecule complementary to said template nucleic acid molecule.

Since the manufacturing method of the present invention uses the DS oligo of the present invention, common descriptions are omitted in order to avoid the complexity of the present specification which causes excessive repeatability.

The application of the present invention using DS oligo provides an improved method for selectively preparing nucleic acid sequences complementary to a target sequence by a template-dependent extension reaction comprising annealing and extending steps. In particular, the preparation of nucleic acid sequences complementary to the target sequence can be achieved by repeating the process of template-dependent extension reactions including annealing, extension and denaturation.

The methods of the invention can be used to prepare nucleic acid molecules that are complementary to template nucleic acid molecules. Such molecules are DNA or RNA. The molecule may be in double or single chain form. If the nucleic acid as a starting material is double stranded, it is preferred to make the two strands in single or partial single chain form. Methods for separating chains include, but are not limited to, heat, alkali, formamide, urea and glycoxal treatment, 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 for achieving the treatments described above are described by 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 carrying out the annealing step. 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). For reverse transcription, oligonucleotide dT primers are used that can hybridize to the poly A tail of mRNA. Oligonucleotide dT primers consist of dTMPs, one or more of which may be replaced with other dNMPs as long as the dT primer can act as a primer. The reverse transcription step can be done with a reverse transcriptase having RNase H activity. In the case of using an enzyme having RNase H activity, an individual RNase H cleavage step can be omitted by carefully selecting reaction conditions.

The method of the present invention does not require that the template nucleic acid molecule have a particular sequence or length. In particular, the molecules may comprise natural prokaryotic nucleic acids, eukaryotic cells (eg, protozoa and parasites, fungi, yeast, higher plants, lower animals and higher animals, including mammals and humans) nucleic acids, viruses (eg, herpes virus) , HIV, influenza virus, Epstein-Barr virus, hepatitis virus, poliovirus, etc.) nucleic acid or non-loid nucleic acid. The nucleic acid molecule also includes nucleic acid molecules that can be chemically synthesized or can be synthesized. Thus, the nucleic acid sequence is either found in nature or not found.

DS oligos used for the present invention hybridize or anneal to one site of the template to form a double chain structure. Suitable annealing conditions for forming this double chain structure 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).

The sequences of the 5'-high T _m specific site and the 3'-low T _m specific site of DS oligo need not have strict complementarity, but only need to be substantially complementary to the sequence to form a stable double chain structure. Thus, deviations from complete complementarity are permitted within the range in which the double-chain structure can be formed. Annealing of DS oligo to a position of template nucleic acid is a prerequisite for template-dependent polymerization by polymerase. Elements paired with bases in nucleic acids where the DS oligo is complementary (see 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)) substantially affect the priming efficiency. The nucleotide composition of the DS oligo affects the temperature at which the annealing is optimized and, ultimately, the priming efficiency.

During the annealing step, the 5'-high T _m specificity sites and 3'-low T _m specificity sites of the DS oligo play an important role as hybridization sites or specificity determining sites (i.e., dual specificity determining sites), while the cleavage sites are hybridized. It does not act as a location and does not combine with the template.

Various DNA polymerases can be used in the extension steps of the methods of the invention, including the “Clenow” fragment 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 is Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus Contains furiosus (Pfu). 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. So that the degree of amplification to a desired joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is desired.

Annealing or hybridization in this method is carried out under stringent conditions that allow for specific binding between the DS oligo and the template nucleic acid. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables. The annealing step in this method is generally carried out under high stringency conditions. However, if the method is applied to a method that requires a mismatch, 5'-high T _m specificity sites and 3'-low T _m specificity sites may be present in the template despite one or more (but limited numbers) of mismatches. It is preferable to carry out the annealing step under stringent conditions of annealing. Such mismatch tolerance is very useful for amplification or detection of genes with genetic variability. Stringent conditions can be readily determined from known criteria.

The annealing step it is advantageous to prevent the annealing of the 3'-low T _m specificity portion but by performing annealing at a higher temperature than that of the 3'-low T _m specificity portion T _m. Preferably, the annealing temperature is the 3'-low T _m specificity portion of the T _m More at least 5 ° C, more preferably at least 10 ° C, even more preferably at least 15 ° C and most preferably at least 20 ° C.

In a preferred embodiment, the annealing temperature is 40-75 ° C, more preferably 45-72 ° C, even more preferably 50-68 ° C, and most preferably 55-65 ° C. Annealing temperatures suitable in the present method are 5'- high T _m specificity portion of the T _m value, and the 3'-low T _m specificity portion of the T _m It can be determined by considering the values independently. That is, the annealing temperature of the method is not determined by the total length and total nucleotide composition of both the 5'-high T _m specific site and the 3'-low T _m specific site, but the 5'-high T _m specific site and / or It is determined by the individual length of the 3'-low T _m specificity site and the nucleotide composition. Usually, the annealing temperature is determined by considering only the T _m value of the 5'-high T _m specific site, which is much higher than the T _m value of the 3'-low T _m specific site, which is optimal.

If mismatch tolerance is required in the annealing step, it is desirable to adjust the annealing temperature to be lower than what is presented above.

The method can be combined with other known procedures to achieve a particular purpose. For example, separation (or purification) of the product produced may be followed by extension. The process can be carried out by gel electrophoresis, column chromatography, affinity chromatography or hybridization. In addition, the amplification products of the present invention can be inserted into suitable cloning vectors. In addition, the production products of the present invention can be expressed in a suitable host having an expression vector.

II. Application to Target Nucleic Acid Sequence Amplification

According to another aspect of the invention, the invention includes amplifying a target nucleic acid sequence using at least two cycles of primer annealing, primer extension and denaturation, using a pair of bispecific oligonucleotides as a primer; The bispecific oligonucleotide comprises a 5'-high T _m specific site, a 3'-low T _m specific site and a split site, wherein the 5'-high T _m specific site is one of the target nucleic acids to hybridize. Has a hybridizing nucleotide sequence that is substantially complementary to position, the cleavage site comprises at least two universal bases, and the 3′-low T _m specificity site is a hybridization nucleotide that is substantially complementary to one position of the target nucleic acid Has a sequence; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under conditions that the 5′-high T _m specific site and the 3′-low T _m specific site are annealed to the target nucleic acid; Annealing in the amplification reaction provides a method for selectively amplifying a target nucleic acid sequence in a DNA or nucleic acid mixture, characterized in that the annealing by the 3'-low T _m specific site alone does not occur.

Since the amplification method of the present invention uses the DS oligo of the present invention, common descriptions are omitted in order to avoid the complexity of the present specification which causes excessive repeatability. In addition, since the method includes an annealing and an extension process, common contents in both methods are omitted in order to avoid the complexity of the present specification which results in excessive repeatability. For example, the composition and structure of the DS oligo used and the conditions for annealing and extension are common between the present method and the method for preparing the nucleic acid molecules described above.

This application using the DS oligo of the present invention provides an improved method of selectively amplifying a target nucleic acid sequence in one nucleic acid or nucleic acid mixture (DNA or mRNA) by performing nucleic acid amplification, preferably PCR (polymerase chain reaction). Can be provided.

An illustrative diagram of selectively amplifying target nucleic acids of double stranded DNA using DS oligos as described above is depicted in FIG. 2. As shown in FIG. 2, a pair of DS oligos are annealed to denatured double stranded DNA templates.

During annealing, the 5'-high T _m specificity site and the 3'-low T _m specificity site play an important role as hybridization sites or specificity determining sites (ie, dual specificity determining sites), while the cleavage sites serve as hybridization sites. Do not and do not combine with the mold. At this time, the cleavage site spatially separates the two terminal sites, that is, the 5'-high T _m specific site and the 3'-low T _m specific site, to form a bubble structure in the DS oligo so that the overall specificity of the DS oligo is double determined. . Details of the subsequent reactions are similar to the conventional primer-associated nucleic acid amplifications known in the art described above.

The method for amplifying a nucleic acid sequence can be performed according to a variety of known primer-associated nucleic acid amplifications. Preferably the method is U.S. It can be carried out according to the PCR process disclosed in patents 4,683,195, 4,683,202 and 4,800,15, more preferably hot start PCR method.

3 shows the selective amplification of the target nucleic acid of mRNA using DS oligo. In the first step, mRNA obtained from various biological samples is reverse transcribed using oligo dT primers and reverse transcriptases that are hybridizable to the poly A tail of the mRNA. The detailed mechanism of reverse transcripts is 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)). Details of the subsequent reactions are similar to those of the known conventional primer-associated nucleic acid amplifications described above.

III. Multiplex DNA  Application to amplification

According to another aspect of the invention, the invention comprises amplifying target nucleotide sequences by performing at least two cycles of primer annealing, primer extension and denaturation using two or more bispecific oligonucleotide pairs as primers, wherein Bispecific oligonucleotides comprise three sites, a 5'-high T _m specificity site, a 3'-low T _m specificity site and a cleavage site, wherein the 5'-high T _m specificity site is one of the nucleotide sequences that hybridizes. Hybridization nucleotide sequences that are substantially complementary to position, wherein the cleavage site comprises at least two universal bases, and the 3′-low T _m specificity site is hybridization that is substantially complementary to one position of the target nucleotide sequence has the nucleotide sequence, the 5'-high T _m Cross portion T _m is higher than the 3'-low T _m specificity portion of the T _m, the cleavage site has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under conditions that the 5′-high T _m specific site and the 3′-low T _m specific site are annealed to the target nucleotide sequence; Annealing in the amplification reaction is carried out under conditions in which no annealing by the 3′-low T _m specificity site alone occurs, amplifying two or more target nucleotide sequences using two or more primer pairs simultaneously in the same reaction. Provide a method.

This application using the DS oligo of the present invention provides an improved method of amplifying one or more target sequences using one or more pairs of primers in the same reaction. In general, it is quite difficult to design multiplex PCR conditions to amplify 10 or more target sequences simultaneously because an optimal PCR reaction is required so that any one specific locus can be amplified without nonspecific byproducts. to be. Since annealing must be performed at a sufficiently high temperature in order for a complete DNA-DNA match to occur in the reaction, the DS oligo of the present invention is ideal for optimizing multiplex DNA amplification because of its ability to improve specificity of amplification. . As used herein, "multiplex PCR" refers to amplifying multiplex DNA targets simultaneously in one polymerase chain reaction (PCR) mixture.

In a particular embodiment of the invention, the multiplexing process comprises performing an amplification reaction comprising performing at least two cycles of primer annealing, primer extension and denaturation using the primer pair of DS oligo, The primer is a bispecific oligonucleotide comprising a 5'-high T _m specific site, a 3'-low T _m specific site and a split site, three sites, wherein the 5'-high T _m specific site hybridizes to the nucleotide sequence Has a hybridizing nucleotide sequence that is substantially complementary to one position of the cleavage site comprises at least two universal bases, and the 3′-low T _m specificity site is substantially complementary to one position of the target nucleotide sequence with hybridization nucleotide sequence, the 5'-high T _m specificity portion of the T _m is the 3'-low T _m higher than the T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under conditions that the 5′-high T _m specific site and the 3′-low T _m specific site are annealed to the target nucleotide sequence; Annealing in the amplification reaction is characterized in that it is carried out under conditions in which annealing by the 3'-low T _m specific site alone does not occur.

Since the present application using the present invention DS oligo is carried out according to the amplification method of the nucleic acid sequence described above except for using one or more target nucleotide sequences and primer pairs, in order to avoid the complexity of the present specification which results in excessive repeatability. The same content between them omits the description. For example, the composition and structure of the DS oligo used and the conditions for amplification are common to the nucleic acid molecule amplification method described above.

According to a preferred embodiment, the annealing temperature is about 40-70 ° C, more preferably 45-68 ° C, even more preferably 50-65 ° C and most preferably 55-65 ° C.

In a preferred embodiment, the products amplified from each of the target nucleotide sequences differ in size for later analysis. According to a preferred embodiment, the amplification product of the multiplex target nucleotide sequence can be analyzed by size discrimination. Size fractional comparisons are carried out through a variety of known methods, ie, electrophoresis and nucleotide sequencing through a polyacrylamide gel matrix or an agarose gel matrix. Nucleotide sequencing can be performed quickly with automated sequencers available from various manufacturers.

As illustrated in the examples below, the multiplexing of the present invention eliminates background problems as well as non-specific problems of known conventional multiplex methods.

The advantage of multiplex amplification is that numerous diseases or specific nucleotide sequence variations (eg, single nucleotide polymorphisms or point mutations) can be analyzed in the same reaction. The number of analytes that can be analyzed at the same time is unlimited; However, the upper limit is about 20, which is determined to depend on the size difference required for the analysis and the method of analyzing the amplified product.

The methods of the present invention can be applied to the diagnosis of genetic and infectious diseases, sex determination, genetic association analysis, and criminal science research.

Ⅳ. DNA  Application to sequencing

The above-described improved specificity is used by DS oligo for direct sequencing as a primer of solution-phase sequencing (especially cycling sequencing) or as a probe of solid-phase sequencing (especially oligonucleotide chip sequencing). Be sure to

According to another aspect of the invention, the invention provides a method of sequencing a target nucleic acid molecule in a DNA or nucleic acid mixture using a bispecific oligonucleotide comprising the following steps:

(a) preparing a nucleic acid molecule complementary to the nucleic acid molecule to be sequenced by performing at least two cycles of primer annealing, primer extension, and denaturation using the bispecific oligonucleotide as a sequencing primer; The bispecific oligonucleotide comprises three sites, a 5'-high T _m specificity site, a 3'-low T _m specificity site, and a cleavage site, wherein the 5'-high T _m specificity site of the target nucleic acid molecule is hybridized. Having a hybridizing nucleotide sequence substantially complementary to one position, said cleavage site comprising at least two universal bases, and said 3'-low T _m specificity site is substantially complementary to one position of said target nucleic acid molecule Has a hybridizing nucleotide sequence; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the target nucleic acid molecule; Annealing in the preparation reaction is carried out under conditions in which annealing by the 3′-low T _m specific site alone does not occur; And

(b) determining the nucleotide sequence of the prepared complementary nucleic acid molecule.

In general, DNA sequencing has been performed by a variety of methods, such as Maksam-Gilbert sequencing, Sanger sequencing, pyro sequencing and exonuclease cleavage sequencing. The sequencing method of the present invention improves pyro sequencing as well as thermal cycle sequencing.

The method can be carried out in accordance with various variations of the Sanger didioxy method. Thermal cycle sequencing of the invention can be performed on PCR amplified nucleic acid templates. Also according to the present invention, thermal cycle sequencing can be performed on nucleic acid templates that are not PCR amplified prior to sequencing.

Briefly, Sanger sequencing is based on the DNA polymerase inserting 2 ', 3'-didioxynucleotides into the nucleic acid chain to terminate the chain reaction (Sanger et al., (1977) PNAS . USA 74: 5463. ). The method invented by Sanger means a didioxy chain termination method. In the most traditional process of this method, DNA segments of sequencing are cloned into single-stranded DNA phages such as M13. When the complementary strand is primed and synthesized by the Klenow fragment of DNA polymerase I, the phage DNA is used as a template. The primer is an oligonucleotide prepared for specific hybridization with the M13 vector region near the 3 'end of the cloned insert. In each of the four sequencing reactions, in the presence of a sufficient amount of didioxy analogs for one of the four deoxynucleotides, primer synthesis is performed, thereby randomizing by insertion of the end-terminal nucleotides. Extension reaction is terminated. Relative concentrations for the dioxy form of didioxy are adjusted to result in a series of termination reactions corresponding to all possible chain lengths that can be analyzed via gel electrointerlocking. The tag inserted in the chain to be synthesized is used to obtain an autoradiogram image of the DNA pattern in each track of electrosynthesis. The sequence of deoxynucleotides in the cloned nucleic acid template is determined by examining the pattern of bands in four lanes.

As a variation of the Sanger method, thermal cycle sequencing methods generally comprise nucleic acid sequencing primers, deoxynucleoside triphosphates, one or more dioxynucleoside triphosphates (ddNTPs), appropriate buffers, heat stable DNA polymerases (eg, Taq polymerase) and the use of a solution comprising the nucleic acid template to be sequenced. For details of heat cycle sequencing, see U.S. Pat. Nos. 5,432,065, 5,723,298, 5,756,285, 5,817,797 and 5,831,06, the contents of which are incorporated herein by reference. The process of the method is generally carried out under thermal cycling conditions similar to common PCR.

A sequencing reaction is performed on the nucleic acid template, followed by analysis of the reaction products to determine the sequence. A significant number of detection methods are known. These methods are Ausubel, FM, etc., Current Protocols in Molecular Biology , (1993) John Wiley & Sons, Inc., New York, generally includes the detection of labels, including radionucleotides, fluorescent, infrared and chemiluminescent labels, and the like.

The label may be labeled with a primer or ddNTP, preferably ddNTP. Most preferred labels are 6-carboxyfluorescein, 6-carboxy-X-rhodamine, 3- (ε-carboxyfetyl) -3'-ethyl-5,5'-dimethyloxacarbocyanine, 6-carboxy-X -Rhodamine, 4,4-difluoro-4-bora-3α, 4α-diaza-s-indacene-3-propionic acid derivatives, and 4,7-dichlororodamine dyes.

Preferably the annealing temperature is 40-70 ° C, more preferably 45-68 ° C and most preferably 50-65 ° C.

The method demonstrates high specific sequencing of the target nucleic acid molecule as shown in the examples below. More specifically, mouse placental-specific homeobox family genes, Psx1 and Psx2 , can be fractionally sequenced using sequencing primers designed to have the unique structure of DS oligos . Considering that the entire sequences of sequencing primers differ only in one base at the 3′-low T _m specificity site, the above-described fractional sequencing can be more clearly understood.

Surprisingly, the present invention allows target nucleic acid molecules contained within genomic DNA or cDNA populations to be sequenced directly without purification or separation. There have been no reports of successful direct sequencing of target nucleic acid molecules within genomic DNA or cDNA populations. When the sequencing method of the present invention is applied to directly sequence target nucleic acid molecules included in cDNA populations obtained from total RNA, the method comprises the following steps:

(a) contacting the population of mRNAs with an oligonucleotide dT primer that hybridizes to the poly A terminus of the mRNA under conditions sufficient for enzymatic deoxyribonucleic acid synthesis of template induction to occur;

(b) reverse transcripting the mRNA hybridized with an oligonucleotide dT primer, the oligonucleotide dT primer generating a primary cDNA chain complementary to the hybridized mRNA;

(c) synthesizing a nucleic acid molecule complementary to the primary cDNA chain to be sequenced using at least two cycles of primer annealing, primer extension, and denaturation using the bispecific oligonucleotide as the sequencing primer; The bispecific oligonucleotide comprises three sites, a 5'-high T _m specificity site, a 3'-low T _m specificity site, and a cleavage site, wherein the 5'-high T _m specificity site of the target nucleic acid molecule is hybridized. Having a hybridizing nucleotide sequence substantially complementary to one position, said cleavage site comprising at least two universal bases, and said 3'-low T _m specificity site is substantially complementary to one position of said target nucleic acid molecule Has a hybridizing nucleotide sequence; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the target nucleic acid molecule; Annealing in the synthesis reaction is carried out under conditions where annealing by the 3′-low T _m specific site alone does not occur; And

(d) determining the nucleotide sequence of the prepared primary cDNA chain.

Ⅴ. Application to Detection of Nucleic Acid Molecules with Genetic Variation

According to another aspect of the present invention, the present invention provides a method for detecting nucleic acid molecules having genetic polymorphism by template-dependent extension of a bispecific oligonucleotide comprising the following steps:

(a) annealing the bispecific oligonucleotide to the template nucleic acid molecule, wherein the bispecific oligonucleotide comprises a 5'-high T _m specific site, a 3'-low T _m specific site and a cleavage site, The 5′-high T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to a position of the template nucleic acid to hybridize, the cleavage site comprises at least two universal bases, and the 3′-low T _m The specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the template nucleic acid, and the annealing is a 3'-low T _m specific site. Annealing by itself does not occur and annealing occurs under conditions that occur when the 5'-high T _m specific site and / or the 3'-low T _m specific site have one or more mismatched bases relative to its target position. Occurs; And

(b) extending said bispecific oligonucleotide to produce said nucleic acid molecule complementary to said template;

(c) detecting whether the template-dependent extension reaction of said bispecific oligonucleotide occurs.

Since the present application using the DS oligo of the present invention is carried out according to the method for preparing nucleic acid sequences described above, common descriptions are omitted in order to avoid the complexity of the present specification which causes excessive repeatability.

The present application using the DS oligo of the present invention can provide an improved method for selectively detecting nucleic acid sequences with genetic variability by template-dependent extension reactions including annealing and extension steps. In particular, detection of target nucleic acid sequences with genetic polymorphism can be achieved by repeating the process of template-dependent extension reactions including annealing, extension and denaturation steps.

The present invention is based on the mismatch tolerance of DS oligos.

Genetic variability has been reported in various genomes. This phenomenon has been regarded as a hindrance to failure-free detection of genes or genomes of interest. The present invention provides a method for solving such conventional problems using DS oligos with mismatch tolerance. DS oligos with a constant sequence can be annealed to several target sequences that show genetic polymorphism, and thus can successfully amplify and detect the desired nucleotide sequence. That is, DS oligo, which was originally invented for the purpose of rapidly improving annealing and hybridization specificity, can also be used in a method requiring mismatch tolerance under conditions in which annealing or stringent conditions are appropriately controlled.

In order to provide a DS oligo that exhibits mismatch tolerance, it is desirable to design the DS oligo based on the conserved region of the identified nucleic acid molecule by aligning all the nucleotide sequences that are obtained.

As used herein, the term “conserved region” refers to a fragment of a nucleotide sequence of a gene or an amino acid sequence of a protein, which is quite similar between various different nucleotide sequences. The term “conserved sequence” Mixed with

In a preferred embodiment, the most conserved sequence in the conserved region is located at the 3'-terminal portion of the DS oligo and the lowest conserved sequence is located at the cleavage site. The 5'-high T _m specificity site and / or the 3'-low T _m specificity site, preferably the 5'-high T _m specificity site, is preferably at least one, preferably one at the target position because of the mismatch tolerance of DS oligo. To three, more preferably one or two mismatched bases. In order to impose mismatch tolerance on the DS oligo, the annealing conditions, in particular the annealing temperature, are important. The annealing 5'-high T _m specificity portion and / or 3'-low T _m specificity portion has one or more if mismatch, 3'-low T _m specificity portion alone, but annealing is not generated in all parts of the It is carried out under the conditions in which annealing by occurs.

Preferably the annealing temperature is about 40-70 ° C, more preferably 45-68 ° C, and most preferably 50-65 ° C.

According to a preferred embodiment, the invention is carried out according to the polymerase chain reaction (PCR).

The detecting step of the method can be carried out by a number of conventional techniques. For example, if the method of the present invention is repeatedly performed to produce enough product to detect on the gel, detection of the template-dependent extension product can be readily carried out by conventional gel electrophoresis. Suitable measurements to detect the occurrence of template-dependent extension reactions, including labels detectable by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electronic and chemical measurements This can be done.

Genetic volatility is most frequently found and generated in viral genome (Nathalie B. et al., ( 2004) Journal of Clinical Microbiology , 42, 3532; Tersa C. et al., (2002) Journal of Infectious Diseases , 185, 1660; Takashi E. et al., (2004) Journal of Clinical Microbiology , 42, 126; and Elizabeth R. et al., (2001) Clinical Infectious Diseases , 32, 1227). Thus, the nucleic acid molecule having the genetic variability to be detected according to the present invention is preferably a nucleic acid of a virus exhibiting the genetic variability. For example, when the present invention is applied to detecting human metapneumoviruses showing genetic variability by PCR, the most preferred primer set designed to have a DS oligo structure is SEQ ID NO: 39 (5 'primer) and SEQ ID NO: 40 (3 'primer) or sequence 39 and 41 (3' primer).

Ⅵ. DS fixed to microarray Application to detection of target nucleotide sequences using oligo

This application is a novel method of detecting target nucleotide sequences by repeating template-dependent reactions on DS oligo-fixed microarrays.

According to another aspect of the invention, the invention provides a method for detecting a target nucleotide sequence in a nucleic acid sample by template-dependent extension of a bispecific oligonucleotide comprising the following steps:

(a) extending a bispecific oligonucleotide as a probe immobilized on a substrate, comprising at least one cycle of hybridization, template-dependent extension and denaturation, wherein said hybridization is effected by contacting the bispecific oligonucleotide with a nucleic acid sample Wherein the bispecific oligonucleotide has a 5′-high T _m specificity site (5′-high T _m specificity portion), 3'-low T _m specificity portion (5'-low T _m a specificity portion and a separation portion, three sites, wherein the 5'-high T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the target nucleotide sequence to hybridize, and The cleavage site comprises at least two universal bases, and the 3′-low T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the target nucleic acid; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a non-base pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the target nucleotide sequence, and the annealing in the amplification reaction is 3'-low. Carried out under conditions in which annealing by the T _m specific site alone does not occur; And

(b) analyzing whether a template-dependent extension reaction has occurred.

An illustrative diagram for detecting target nucleotide sequences in nucleic acid samples using DS oligo-fixed microarrays is shown in FIG. 4.

The method using DS oligo can be carried out under suitable hybridization conditions as determined by the optimization procedure. 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 oligonucleotides and the target nucleotide sequence. For example, low stringency conditions are preferred when relatively short oligonucleotides are used. 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, NY (2001); And MLM Anderson, Nucleic Acid Hybridization , Springer-Verlag New York Inc. See NY (1999).

DS oligo is immobilized on the substrate. Preferred substrates include suitable solid or semi-solid supports such as membranes, filters, chips, slides, water, fever, magnetic or nonmagnetic beads, gels, tubing, plates, macromolecules, microparticles and capillary tubes. Immobilization is accomplished by chemical reaction or covalent bonds by ultraviolet light. In an embodiment of the invention, the DS oligo is bound to a glass surface or polylysine-coated surface modified to include an epoxy compound or an aldehyde group. DS oligo is also bound to the substrate via linkers (eg ethylene glycol oligomo and diamine). By conventional manufacturing techniques such as photolithography, inkjetting, mechanical microspotting and similar methods, DS oligos can be fabricated into arrays or arrays.

According to the method, the dNTPs used in the extension step are preferably labeled. For labeling, substances detectable by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electronic and chemical measurements are used. For example, labels include, but are not limited to, radioisotopes such as P ³² and S ³⁵ , chemiluminescent compounds, spectroscopic markers such as fluorescent markers and dyes, and magnetic labels. For example, dyes include, but are not limited to, quinoline dyes, triarylmethane dyes, phthaleins, azo dyes and cyanine dyes. Fluorescent markers include, but are not limited to, fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia). Labeling is performed according to various known methods.

After hybridization of the DS oligo as a probe immobilized on a substrate, preferably on a solid support, with the target nucleotide sequence of the nucleic acid sample, the DS oligo hybridized with the target nucleotide sequence is followed by dNTPs, preferably fluorescence-labeled, in a template-dependent step. is extended using dNTPs and DNA polymerase. It is preferable to perform step (a) repeatedly so that all or most of the DS oligos hybridize with the target nucleotide sequence, so that a more reproducible hybridization analysis result can be obtained.

The occurrence of hybridization is confirmed by various known methods depending on the type of label used. For example, fluorescence microscopy, preferably confocal fluorescence microscopy, is used to detect fluorescent labels, and the intensity of the signal detected with such a device increases in proportion to the degree of hybridization. In general, a fluorescence microscope is attached with a scanning device for forming a quantitative two-dimensional image of hybridization intensity. The intensity of the signal detected by such a device is proportional to the degree of hybridization and the degree of template-dependent extension.

The invention is illustrated in more detail by the examples. The following examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

1A and 1B show the principle of the dual specificity (DS) oligonucleotides of the present invention in a template-dependent extension reaction. 1A shows high hybridization specificity of DS oligonucleotides under high stringency conditions. 1B shows mismatch tolerance of DS oligonucleotides.

Figure 2 shows the process of selectively amplifying target nucleic acids of double-stranded DNA using the DS oligonucleotide primer of the present invention.

Figure 3 shows the process of selectively amplifying the target nucleic acid of the mRNA using the DS oligonucleotide of the present invention.

4 shows a template-dependent extension reaction to selectively determine target nucleic acids using bispecific oligonucleotides on oligonucleotide microarrays.

Figure 5 shows the cytokine family genes IL-1beta and IL-19 in the conventional primer set (IL-1b-5'-0 and IL-1b-3'-0, lane 1; IL-19-5'-0 And IL-19-3'-0, lane 3) and bispecific oligonucleotides (IL-1b-5 'and IL-1b-3', lane 2; IL-19-5 'and IL-19-3', Agarose gel photograph showing the results of PCR amplification using lane 4).

6A is agarose gel photograph showing the results of 3'-RACE (fast amplification of cDNA ends) of DEG 10 gene demonstrating PCR specificity of bispecific oligonucleotides. Lane 1 is for primers with a perfect match sequence, lanes 2-4 are 3'-low T _m For primers with 3, 2 and 1 base mismatches at specific sites, respectively, lanes 5-7 are 5'-high T _m For primers with 5, 3 and 2 base mismatches, respectively, at specific sites. M is a 100-bp sized marker produced by a Forever 100-bp Ladder Personalizer.

6B is an agarose gel photograph showing the 3′-RACE results of the DEG 10 gene demonstrating mismatch tolerance of bispecific oligonucleotides in PCR. Lane 1 is for one primer with a perfect match sequence, lanes 2-4 are 3′-low T _m For primers with 3, 2 and 1 base mismatches at specific sites, respectively, lanes 5-7 are 5'-high T _m For primers with 5, 3 and 2 base mismatches, respectively, at specific sites. M is a 100-bp sized marker produced by a Forever 100-bp Ladder Personalizer.

Figure 7A shows the sequence of 5 'primers for the 3' race of the mouse placental-specific homeobox family genes, Psx1 and Psx2 . Psx1-5'-40 and Psx2-5'-40 are conventional primers and Psx1-5'-41 and Psx2-5'-41 are primers designed by the present invention.

7B is an agarose gel photograph showing the results of 3'-RACE of Psx1 and Psx2 . Lane 1 is 3'-RACE of Psx1 using the bispecific oligonucleotide primer Psx1-5'-41; Lane 2 is 3'-RACE of Psx2 using bis specific oligonucleotide primer Psx2-5'-41; Lane 3 is 3'-RACE of Psx1 using conventional primers Psx1-5'-40; And lane 4 is the result of 3'-RACE of Psx2 using the conventional primer Psx2-5'-40. M is a 100 bp sized marker produced by a Forever 100 bp Ladder Personalizer.

8 shows bispecific oligonucleotide primers Psx1-5'-41 ( Psx1 If) and Psx2-5'-41 (Psx2 case) for use as a sequencing primer in mouse placenta cDNA pool Psx1 Wow Direct cycling sequencing of the Psx2 gene is shown.

9 shows multiplex PCR results using nine sets of cytokine family gene-specific bispecific primers. Lane 1 comprises multiplex PCR for nine cytokine genes; Lane 2 was monoplexed PCR for IL-3 (200 bp); Lane 3 was monoplexed PCR for IL-15 (250 bp); Lane 4 was monoplexed PCR for IL-18 (300 bp); Lane 5 was monoplexed PCR for IL-25 (350 bp); Lane 6 was monoplexed PCR for IL-2 (400 bp); Lane 7 shows monoplex PCR for IL-6 (450 bp); Lane 8 shows monoplex PCR for IL-19 (500 bp); Lane 9 shows monoplex PCR for IL-1beta (550 bp); And lane 10 shows the result of monoplex PCR for IL-10 (600 bp). M is a 100-bp sized marker produced by a Forever 100-bp Ladder Personalizer.

Figure 10 shows the results of PCR amplification of the human metapneumovirus (hMPV) fusion glycoprotein (F) gene using a bispecific oligonucleotide primer. Target PCR using lane 1, hMPV5'-585 and hMPV 3'-689 primer sets; Target PCR using lane 2, hMPV5'-585 and hMPV 3'-1007 primer sets; Lane 3, target PCR using human beta-actin primers; Lane 4, target PCR without template; Lane 5, target PCR without template.

Example  1: dual specificity ( DS ) Oligonucleotides  Used PCR  Specificity

In order to amplify target nucleotide sequences of mouse cytokine family genes, IL-19 and IL-1 beta, DS oligonucleotides developed by the present invention were used as primers. Amplification procedures and results of target nucleotide sequences of IL-19 and IL-1 beta using DS primers are described.

The following conventional primer sequences were selected and used to compare the PCR specificity with DS oligonucleotides:

IL-19 specific conventional primers used in the Examples are as follows (500 bp):

IL19-5'-0 5'-GTCTCATCTGCTGCCCTTAAGTCTCTAGGAGAACT-3 '(SEQ ID NO: 1); And

IL19-3'-0 5'-CATAGGCCTGGAAGAAGCCGCTTTACAATAAGTTAG-3 '(SEQ ID NO: 2).

IL-1 beta specific conventional primers used in the Examples are as follows (550 bp):

IL1b-5'-0 5'-GGAGAGTGTGGATCCCAAGCAATACCCAAAGAAG-3 '(SEQ ID NO: 3); And

IL1b-3'-0 5'-AGACCTCAGTGCAGGCTATGACCAATTCATCCC-3 '(SEQ ID NO: 4).

Inventive DS oligonucleotides were applied to conventional primer sequences to verify whether DS oligonucleotides could solve major problems resulting from conventional primer sequences such as background and nonspecific product generation.

DS primers contain the same sequence as conventional primers, except for the cleavage site having a polydeoxyinosine [poly (dI)] linker between the 5 ′ and 3 ′ sites. DS primer is 5'- high T _m T _{m at} specific site is 3'-low T _m Nopeumyeonseo than the T _m specificity portion are designed to have a T _m of the cleavage site with the lowest T _m among the three regions.

DS primers for IL-19 used in the Examples are as follows (500 bp):

IL19-5 '5'-GTCTCATCTGCTGCCCTTAAIIIIITAGGAGAACT-3' (fifth sequence); And

IL19-3 '5'-CATAGGCCTGGAAGAAGCCGIIIIICAATAAGTTAG-3' (sixth sequence). Wherein I is deoxyinosine.

DS primers for IL-1 beta used in the Examples are as follows (550 bp):

IL1b-5 '5'-GGAGAGTGTGGATCCCAAGCIIIIICCAAAGAAG-3' (seventh sequence); And

IL1b-3 '5'-AGACCTCAGTGCAGGCTATGIIIIITTCATCCC-3' (eighth sequence). Wherein I is deoxyinosine.

2 μl of genome DNA isolated from placental tissue of ICR mice, 2 μl of 10 × PCR buffer containing 15 mM MgCl ₂ (Roche), 2 μl of dNTP (2 mM each of dATP, dCTP, dGTP and dTTP), 5 Target PCR at a 20 μl final volume containing 1 μl of DS or conventional primer (10 μM), 1 μl of 3′DS or conventional primer (10 μM), and 0.5 μl of Taq polymerase (5 units / μL; Roche) Amplification was performed; The tube containing the reaction mixture was placed in a preheated (94 ° C.) heat cycler; The sample was denatured at 94 ° C. for 5 minutes, repeated 1 minute at 94 ° C., 1 minute at 60 ° C. and 1 minute at 72 ° C., and then treated at 72 ° C. for 7 minutes.

The amplified product was analyzed by electrophoresis on 2% agarose gel and stained with EtBr (ethidium bromide). PCR products were detected by non-radioactive detection methods such as self-radiation or silver staining (Gottschlich et al., (1997) Res . Commun . Mol . Path . Pharm . 97, 237-240; Kociok , N., et. al . (1998) Mol . Biotechnol . 9, 25-33 ), or methods using fluorescently labeled oligonucleotides (Bauer D., et al., (1993) Nucleic Acids Res . 21, 4272-4280; Ito, T. et al., (1994) FEBS Lett . 351, 231-236. Luehrsen, KR et al., (1997) BioTechniques 22, 168-®; Smith, NR et al., (1997) BioTechniques 23, 274-279) And methods using biotinylated primers (Korn, B. et al., (1992) Hum . Mol . Genet . 1, 235-242; Tagle, DA et al., (1993) Nature 361. 751-753; Rosok, O. et al., (1996) BioTechniques 21, 114-121) can be detected on modified polyacrylamide gels.

As shown in Figure 5, the cytokine family genes IL-1b and IL-19 for IL-1b-5 'and IL-1b-3', and IL-19-5 'and IL-19-3' each primer Target PCR amplification using the set resulted in a single band corresponding to the expected size of 550-bp for IL-1 beta (lane 2) and 500-bp for IL-19 (lane 4), respectively. Cloning and sequencing of the clones then confirmed that the bands were fragments of IL-1 beta and IL-19. In contrast, the conventional primer set (IL19-5'-0) that did not contain [Poly (dI)] IL19-3'-0;IL1b-5'-0 and IL1b-3'-0) produced nonspecific products (FIG. 5, lane 1 and lane 3). The result is three different T _m Designed to have sites (5'-high T _m specific sites, 3'-low T _m specific sites and cleavage sites), DS primers overcome the problems caused by conventional primer sequences such as the generation of backgrounds and nonspecific products It shows that specificity can be greatly improved.

Example  2: double specificity ( DS ) Oligonucleotides  Used PCR  Specificity assessment

The bispecific oligonucleotides of the present invention are characterized by high hybrid specificity and mismatch tolerance by their unique structure, which depends on the hybridization stringency. The high hybrid specificity of DS oligonucleotides is achieved under high stringency conditions in which both 5′- and 3′-sites are annealed to the template. On the other hand, mismatch tolerance of DS oligonucleotides is made under strict conditions where both 5'- and 3'- sites anneal to the template, despite the presence of one or more base pair mismatches that are not limited.

DEG10 (Kim, YJ et al., (2004) Annealing control primer system for identification of differentially expressed genes on agarose gels. BioTechniques 36: 424-434; In the 3'-RACE experiment of XM_129567), the dual specificity of the DS oligonucleotide developed according to the present invention was evaluated in terms of hybrid specificity and mismatch tolerance. For this evaluation, several nucleotides at both sites were replaced with other nucleotides to mismatch the target template sequence.

The 5′-DEG10-specific DS primers used in the examples are as follows:

DEG10-5'-108: 5'-TGTAGTTTTGGGTTTCCTCCIIIIICTCCGATG-3 '(ninth sequence);

DEG10-5'-103: 5'-TGTAGTTTTGGGTTTCCTCCIIIIICT G C C AT C -3 '(tenth sequence);

DEG10-5'-102: 5'-TGTAGTTTTGGGTTTCCTCCIIIIICTCC C AT C- 3 '(SEQ ID NO: 11);

DEG10-5'-101: 5'-TGTAGTTTTGGGTTTCCTCCIIIIICTCC C ATG-3 '(twelfth sequence);

DEG10-5'-158: 5'-TGTA C TT A TG C GT A TC G TCCIIIIICTCCGATG-3 '(SEQ ID NO: 13);

DEG10-5'-138: 5'-TGTA C TTTTG C GTTTC G TCCII III CTCCGATG-3 '(SEQ ID NO: 14); And

DEG10-5'-128: 5'-TGTAGTT A TGGGT A TCCTCCIIIIICTCCGATG-3 '(15th sequence).

Substituted nucleotides are shown underlined and bolded, and I represents deoxyinosine.

A. high Strict conditions  Under PCR  Increased specificity

To synthesize primary cDNA chains by reverse transcriptase as described above, total RAN was isolated and used from 17.5-dpc (E17.5) placental tissue of ICR mice (Hwang, IT, et al., (2003) Annealing control primer system for improving specificity of PCR amplification BioTechniques 35:. 1180-1184). The total RNA was subjected to reverse transcription for 20 hours at 42 ° C. in a reaction volume of 20 μl prepared as follows: 3 μl of total RNA, 4 μl of 5 × buffer (Promega, USA), 5 μl of dNTPs (2 mM each), 2 μl of 10 μM cDNA synthesis primer (oligo (dT) _20- Adapter), 0.5 μl of RNase inhibitor (40 units / μl, Promega), and 1 μl of reverse transcriptase (200 units / μl, Promega). 180 μl of ultra-purified H ₂ 0 was added to dilute the primary cDNA chain. Oligo (dT) ₁₈ , a cDNA Synthesis Primer -ACP1 is _{5'-CTGTGAATGCTGCGACTACGATIIIII (T) 18- 3} ' , where I is deoxy inosine.

2 μl of diluted primary cDNA chain (30 ng), 2 μl of 10 × PCR buffer containing 15 mM MgCl ₂ (Roche), 2 μl of dNTP (2 mM each of dATP, dCTP, dGTP and dTTP), DEG10-specific 3'-RACE of DEG10 at a final volume of 20 μl containing 1 μl of red DS primer (10 μM), 1 μl of oligo (dT) _15- ACP2 (10 μM) and 0.5 μl of Taq polymerase (5 unit / μl; Roche) Was conducted; The tube containing the reaction mixture was placed in a preheated (94 ° C.) heat cycler; The sample was denatured at 94 ° C. for 5 minutes, followed by 1 minute at 94 ° C., 1 minute at 68 ° C., 1 minute at 72 ° C., and then incubated at 72 ° C. for 7 minutes. Oligo (dT) ₁₅ -ACP2 is 5'-CTGTGAATGCTGCGACTACGATIIIII (T) 15-3 'where I is deoxyinosine.

B. DS Of oligonucleotides Mismatch  permit

Sample DS primers, templates and PCR conditions for performing 3'-RACE of DEG10 were the same as in Example 2A, and the annealing temperature was varied. PCR amplification was carried out under the following conditions: 5 min at 94 ° C., 3 min at 60 ° C. and 3 min at 72 ° C .; Then repeat the procedure 40 times at 94 ° C., 1 minute at 65 ° C. and 40 seconds at 72 ° C. for 29 repeats and a final extension cycle for 7 minutes at 72 ° C.

As a result, Figure 6A shows the high hybrid specificity of DS oligonucleotide primers in the 3'-RACE experiment of DEG10. 5′-DEG10-specific DS primers (DEG10-5′-108) produced the 677 bp product expected in DEG10 3′-RACE (lane 1). Conversely, other primers with mismatch sequences at the 5 'or 3' site (DEG10-5'-103, DEG10-5'-102, DEG10-5'-101, DEG10-5'-158, DEG10-5) '-138 and DEG10-5'-128) did not produce any products: mismatches of three (lane 2), two (lane 3), or one (lane 4) base at the 3' site; 5 '(lane 5), 3 (lane 6), or 2 (lane 7) base mismatches in the 5' region.

These results demonstrate that the mismatched bases can be distinguished at the 3'-end as well as the 5'-end under stringent conditions with high bispecificity of the DS primer.

Generally, the primer site that should be perfectly complementary to the template is the 3'-end, since this end is the site extended by the DNA polymerase and is therefore the most important site for annealing to the correct target sequence. to be. The 5'-terminus of the primer, on the other hand, may be modified to have additional sequences, such as restriction sites and promoter sequences, which are less important in determining the specificity of annealing to the target sequence and are not complementary to the template ( McPherson, MJ, Moller, SG ( 2000) PCR. BIOS Scientific Publishers,. Springer-Verlag New York Berlin Heidelberg, NY.). In contrast, the particular advantage of DS primers manifested by the dual specificity due to the unique structure allows to distinguish mismatched bases at the 5'-end as well as at the 3'-end.

6B shows an example of mismatch tolerance of DS oligonucleotides as 3′-RACE of DEG10. DS primers (DEG10-5'-108, DEG10-5'-101, DEG10-5'-128, and DEG10-5'-138), even if there are no or few mismatched nucleotides at the 5'- or 3'-terminus Still produced the expected 677 bp product of DEG10 3′-RACE (lanes 1,4,6 and 7). Whereas other primers with more mismatched nucleotides in the 5 'or 3' region (DEG10-5'-103, DEG10-5'-102, DEG10-5'-158) did not produce any product (lane 2, 3 and 5). The results indicate that DS primers can be used to amplify various nucleotide sequences that require mismatch tolerance.

In summary, the results support the following principle of DS primers:

1) Only when annealing occurs at both the 5′-high Tm specific site and the 3′-low Tm specific site, the DS primer is extended to synthesize nucleic acid molecules complementary to the template (lane 1); But,

2) when annealing occurs at only 5'-high Tm specific sites, not at 3'-low Tm specific sites, the DS primers are not extended to synthesize nucleic acid molecules complementary to the template (lanes 2-4); And

3) Even when the sequence of the 3'-low Tm specificity site matches perfectly with the template, the annealing does not occur with the 3'-low Tm specificity site alone under high stringency conditions (lanes 5-7). With regard to the annealing site, the DS primer is clearly different from the annealing control primer (ACP), which extends when only the 3'-terminal site is annealed in the initial PCR step (Hwang, IT, et al., (2003) Annealing control primer system for improving specificity of PCR amplification. BioTechniques 35: 1180-1184).

Example  3: double specificity ( DS ) Oligonucleotides  Single base distinction

To demonstrate the dual specificity of DS oligonucleotides for single base distinction, cDNAs of mouse placental specific homeobox family genes, Psx1 and Psx2 , were amplified using either conventional primers or DS primers. Two Psx Total sequence homology between cDNAs was 91% at the nucleotide level (Han, YJ, et al., (2000) Identification and characterization of Psx2 , a novel member of the Psx (placenta-specific homeobox) family. Gene 241: 149-155. Psx1 and Psx2 differ by one or two base differences 5'-primers were designed to be distinguished (FIG. 7A). However, the 3'-primers were designed to have conserved sequences of both Psx cDNAs. Psx1 - and Psx2 - specific conventional and DS primer sequences are:

Psx1-5'-10: 5'-AAGGAAGACATGCTGGTGATGGTGCTTCT A GC T- 3 '(SEQ ID NO: 16);

Psx2-5'-10: 5'-AAGGAAGACATGCTGGTGATGGTGCTTCT G GC C- 3 '(seventh sequence);

Psx1-5'-11: 5'-AAGGAAGACATGCTGGTGATIIIIITTCT A GC T- 3 '(SEQ ID NO: 18);

Psx2-5'-11: 5'-AAGGAAGACATGCTGGTGATIIIIITTCT G GC C- 3 '(19 th sequence);

Psx1-5'-40: 5'-TCTTGCACGATGGATGGGTGTGGATGAAT G TGA-3 '(20th sequence);

Psx2-5'-40: 5'-TCTTGCACGATGGATGGGTGTGGATGAAT C TGA-3 '(21st sequence);

Psx1-5'-41: 5'-TCTTGCACGATGGATGGGTGIIIIIGAAI G IGA-3 '(22nd sequence);

Psx2-5'-41: 5'-TCTTGCACGATGGATGGGTGIIIIIGAAI C IGA-3 '(SEQ ID NO: 23); And

Psx-3'-2: 5'-TTCATCCACACCCATCCATCIIIIIAGATCCCT-3 '(SEQ ID NO. 24),

Wherein Psx1 - or Psx2 - specific nucleotides are shown in bold and underlined.

A. Synthesis of Primary cDNA Chains

The primary cDNA chain of the mouse placenta synthesized in Example 2 was used as a starting material for target PCR of 3'-RACE and Psx cDNA.

B. Psx1 - and Psx2 - Specific DS 3 ' RACE of Psx1 and Psx2 with Primer

2 μl of diluted primary cDNA chain (30 ng), 2 μl of 10 × PCR buffer (Roche) containing 15 mM MgCl ₂ , 2 μl of dNTP (2 mM each of dATP, dCTP, dGTP and dTTP), 5′- Psx1 end including; (Roche 5 unit / ㎕) 0.5 ㎕ - - and 5'- Psx2-specific DS or conventional primer (10 μM) 1 ㎕, oligo (dT) 15 -ACP2 (10 μM) 1 ㎕ and Taq polymerase 3'-RACE of Psx1 and Psx2 was performed in 20 μl volume; A tube containing the reaction mixture was placed in a preheated (94 ° C.) heat cycler; The sample was denatured at 94 ° C. for 5 minutes, then repeated 30 minutes for 1 minute at 94 ° C., 1 minute at 60-65 ° C., and 1 minute at 72 ° C., and then incubated at 72 ° C. for 7 minutes.

C. Psx1 -and Psx2 - Specific DS Psx1 using primers and Psx2 Target Nucleic Acid Amplification

2 μl of diluted primary cDNA chain (30 ng), 2 μl of 10 × PCR reaction buffer (Roche) with 15 mM MgCl ₂ , 2 μl of dNTP (2 mM each of dATP, dCTP, dGTP and dTTP), 5′- Psx1 - or 5'- Psx2 - specific DS primer or one of the conventional primer (10 μM) 1 ㎕, Psx -3'-2 (10 μM) 1 ㎕ and Taq polymerase (5 unit / ㎕; Roche) 0.5 ㎕ Target PCR amplification of Psx1 and Psx2 was performed in a final volume of 20 μl including The tube containing the reaction mixture was placed in a preheated (94 ° C.) thermal cycler; The sample was denatured at 94 ° C. for 5 minutes, then repeated 30 minutes for 1 minute at 94 ° C., 1 minute at 60-65 ° C., and 1 minute at 72 ° C., and then incubated at 72 ° C. for 7 minutes.

In conclusion, FIG. 7B shows that 5 ' Psx1 -or Psx2 -specific fries produce the product of 3'-RACE and target PCR. Since the two Psx cDNAs are different from each other by removing or adding 29 bp at the 3'-end side, it can be expected that the products having a difference of 29 bp are amplified by 3'-RACE. Psx1 - or Psx2 - specific DS primer Psx1-5'-41 and the 3'-RACE of Psx1 cDNA using the Psx2-5'-41 are each expected to be 311 bp (lane 1) and 282 bp (lane 2) Size Generated a single band matching. Through subsequent sequencing of the product generated by 3'-RACE, the 5'- Psx1 -and- Psx2 -specific primers were Psx1 and Psx2, respectively. It was confirmed that each cDNA was amplified. In contrast, conventional primers (Psx1-5'-40 and Psx2-5'-40) that do not follow the principles of DS oligonucleotides did not differentiate between the two Psx cDNAs (lane 3 and lane 4).

The results show that the DS primers according to the invention can distinguish one single base mismatch. Therefore, to identify point mutations or genotyping single nucleotide polymorphisms (SNPs), the DS oligonucleotides of the present invention can be applied.

Example 4 Direct Sequencing of Target cDNA in a cDNA Pool Using Bispecific ( DS ) Oligonucleotides

Most attempts to identify and isolate new cDNA result in obtaining clones that represent only a portion of the mRNA sequence. Once the partial sequence has been identified, the remainder of the transcript is often obtained through PCR-based methods such as typical cDNA library screening or RACE (cDNA terminal fast amplification), and then sequencing the resulting cDNA. All current methods are therefore an essential step in obtaining sequence information for the rest of the transcript. If missing sequence information is obtained directly from cDNA fragmentation from target cells, this time-consuming essential process can be completely avoided and the target cDNA sequence can be determined directly from a crude biological sample. have.

In order to directly sequence the cDNA of the mouse placental specific homeo box gene Psx using the placental primary cDNA chain pool, the DS oligonucleotide of the present invention was used as a primer. Methods and results of direct sequencing of placental specific gene cDNA from placental cDNA pools are described here. The same 5'-Psx specific DS primers used in Example 3 were used. These primers are Psx 1-5'-11, Psx 2-5'-11, Psx 1-5'-41 and Psx 2-5'-41.

A. Primary cDNA Chain Formation

Psx The primary cDNA chain of the mouse placenta synthesized in Example 2 was used as a template for direct sequencing of cDNAs.

B. Psx Specific DS Psx in placental cDNA pool using primers Direct Sequencing of cDNA

13 μl (150 ng) of diluted primary cDNA chain, 2 μl of ABI PRISM Big Dye Terminator reaction mixture (Applied Biosystems, USA), 2 μl of 5 × sequence buffer (Appled Biosystems) and 5′- Psx1 − or 5′- Psx2 A cycle sequencing reaction was performed at 20 μl of the final volume containing 1.6 μl of one of the specific DS primers (1 μM); The tube containing the reaction mixture was placed in a preheated (94 ° C.) heat cycler; The sample was denatured at 94 ° C. for 5 minutes and then repeated 40-50 times for 10 seconds at 94 ° C., 3 minutes at 50-60 ° C. and 4 minutes at 60-65 ° C. Sequencing products were purified as follows. 1) Add 2 μl of 3 M sodium acetate (pH 4.6) and 50 μl of fresh and cold 100% EtOH, 2) hold at −75 ° C. for 30 minutes, 3) centrifuge at 13,000 g for 15-30 minutes and supernatant Remove, 4) wash with 200 μl of 70% EtOH, 5) centrifuge at 13,000 g for 15-30 minutes and carefully remove supernatant and dry. The pellet was resuspended in 10 μl HiDi formamide immediately before sequencing products were analyzed on an ABI PRISM 3100 genetic analyzer.

Surprisingly, Psx DS primers correctly sequenced their specific Psx cDNAs. That is, Psx1 specific DS primer (Psx1-5'-41) only sequenced Psx1 cDNA, and Psx2 specific DS primer (Psx2-5'-41) was only Psx2 Only cDNA was sequenced (FIG. 8). The 29 bp deletion region in Psx2 is indicated by black bars. In contrast, conventional primers (Psx1-5'-40 and Psx2-5'-40) that do not follow the principles of DS oligonucleotides did not distinguish between the two Psx cDNAs.

These results indicate that DS primers can distinguish single base mismatches not only in PCR amplification but also in cycle sequencing.

Example  5: DS Oligonucleotides  Multiplex with multiplex ) PCR

To demonstrate the application of DS oligonucleotide primers in multiplex PCR, nine different cytokine family genes were amplified with DS primers. Methods and results of multiplex PCR amplification using DS primers are described herein. Cytokine family gene-specific DS primers were designed to generate a 50 bp ladder using the longest exon sequence of each cytokine gene.

DS primers for IL-3 used in the Examples are as follows (200 bp):

IL3-5 '5'-GCTGCCAGGGGTCTTCATTCIIIIICTGGATGA-3' (25th sequence); And

IL3-3 '5'-GGCCATGAGGAACATTCAGAIIIIIGGTGCTCT-3' (stage 26).

DS primers for IL-15 used in the Examples are as follows (250 bp):

IL15-5 '5'-ATGTAGCAGAATCTGGCTGCIIIIIATGTGAGG-3' (27th sequence); And

IL15-3 '5'-ATGTGATCCAAGTGGCTCATIIIIICCTTGTTAGG-3' (SEQ ID NO: 28).

DS primers for IL-18 used in the Examples are as follows (300 bp):

IL18-5 '5'-AGGAAATGGATCCACCTGAAIIIIITGATGATATA-3' (SEQ ID NO. 29); And

IL18-3 '5'-ATGGAAATACAGGCGAGGTCIIIIIAAGGCGCA-3' (30th sequence).

DS primers for IL-25 used in the Examples are as follows (350 bp):

IL25-5 '5'-AGCTCTCCAAGCTGGTGATCIIIIICAAGGCGG-3' (31st sequence); And

IL25-3 '5'-GAGCTGCCCTGGATGGGGTTIIIIIGTGGTCCT-3' (32nd sequence).

DS primers for IL-2 used in the Examples are as follows (400 bp):

IL2-5 '5'-CTCTGACAACACATTTGAGTGCIIIIICGATGATGAG-3' (SEQ ID NO: 33); And

IL2-3 '5'-GTGCTGTCCTAAAAATGACAGAIIIIIGAGCTTATTT-3' (34 th sequence).

DS primers for IL-6 used in the Examples are as follows (450 bp):

IL6-5 '5'-CCAATGCTCTCCTAACAGATAAIIIIIAGTCACAGAA-3' (35th sequence); And

IL6-3 '5'-AGGTAAACTTATACATTCCAAGAAAIIIIITGGCTAGG-3' (36th sequence).

DS primers for IL-19 used in the Examples are as follows (500 bp):

IL19-5 '5'-GTCTCATCTGCTGCCCTTAAIIIIITAGGAGAACT-3' (fifth sequence); And

IL19-3 '5'-CATAGGCCTGGAAGAAGCCGIIIIICAATAAGTTAG-3' (sixth sequence).

DS primers for IL-1 beta used in the Examples are as follows (550 bp):

IL1b-5 '5'-GGAGAGTGTGGATCCCAAGCIIIIICCAAAGAAG-3' (seventh sequence); And

IL1b-3 '5'-AGACCTCAGTGCAGGCTATGIIIIITTCATCCC-3' (eighth sequence).

DS primers for IL-10 used in the Examples are as follows (600 bp):

IL10-5 '5'-AAGGCCATGAATGAATTTGAIIIIITCATCAACTG-3' (Sequence 37); And

IL10-3 '5'-TGACAGTAGGGGAACCCTCTIIIIIGCTGCAGG-3' (38th Sequence)

A. Cytokine Family Gene-Specific DS Monoplex PCR with a set of primers

10 μl PCR buffer containing 2 μl (50 ng) of mouse genome DNA, 15 mM MgCl ₂ (Roche) 2 μl, dNTP (2 mM each of dATP, dCTP, dGTP and dTTP), 1 μl of each cytokine family gene-specific 5′DS primer (10 μM), Taq polymerase (5 unit / μl; Roche) single target PCR amplification for each cytokine family gene in a final volume of 20 μl, including 0.5 μl; The tube containing the reaction mixture was placed in a preheated (94 ° C.) thermal cycler; The sample was denatured at 94 ° C. for 5 minutes, followed by 1 minute at 94 ° C., 1 minute at 60-65 ° C., 1 minute at 72 ° C., and then incubated at 72 ° C. for 7 minutes.

B. Cytokine Family Gene Specific DS Multiplex PCR with 9 sets of primers

Multiplex PCR amplification was performed in a single tube using 9 sets of cytokine family gene specific DS primers; Mouse genome DNA 100 ng, 5 μl 10 × PCR reaction buffer (Roche) with 15 mM MgCl ₂ , 5 μl dNTP (2 mM each of dATP, dCTP, dGTP and dTTP), each cytokine family gene-specific 5′DS Final volume 50 μl, containing 1 μl primer (0.2-5 μM), 1 μl of each cytokine family gene-specific 3′DS primer (0.2-5 μM) and 0.5 μl Taq polymerase (5 unit / μl; Roche) A reaction mixture was prepared; The tube containing the reaction mixture was placed in a preheated (94 ° C.) heat cycler; PCR conditions were performed once 5 minutes at 94 ℃, 3 minutes at 50 ℃ and 3 minutes at 72 ℃ once; The procedure is 40 repetitions at 94 ° C., 1 minute at 60 ° C., and 40 seconds at 72 ° C. for 29 repetitions and a final extension cycle of 5 minutes at 72 ° C.

As shown in Figure 9, multiplex PCR amplification produces several bands that match the expected sizes from 200 bp to 600 bp for nine different cytokine gene products (Figure 4, lane 1). Each monoplex PCR amplification was performed at 200 bp for IL-3 (FIG. 4, lane 2), 250 bp for IL-15 (FIG. 4, lane 3), 300 bp for IL-18 (FIG. 4, lane 4), IL. 350 bp for FIG. 25 (FIG. 4, lane 5), 400 bp for IL-2 (FIG. 4, lane 6), 450 bp for IL-6 (FIG. 4, lane 7), 500 bp for IL-19 (FIG. 4) , Lane 8), 550 bp for IL-1 beta (FIG. 4, lane 9) and 600 bp for IL-10 (FIG. 4, lane 10) to generate single bands matching the expected sizes, respectively.

Therefore, it can be seen that the DS primer developed by the present invention can be successfully applied to multiplex PCR. The unique structure of DS oligonucleotides solves the common problems of conventional multiplex PCR, namely primer interference and dimmer formation.

Example 6: Bispecific ( DS ) Oligonucleotides Human meth pneumophila virus (metapneumovirus) Detection of mismatches allowed (tolerance)

To demonstrate the application of DS oligonucleotide primers in mismatch tolerance, DS primers were used to detect human metapneumovirus (hMPV) in diagnostic samples. The method and results of detecting human metapneumovirus (hMPV) using DS primers are described here. This example should not be construed to limit the application of the invention to detecting a particular virus.

DS primers were designed based on the conserved region of the fusion glycoprotein (F) gene obtained by aligning the sequences of all hMPV isolates (see Table 1). To allow for genetic diversity of these isolates, primers were designed according to the following criteria: (a) the conserved region should be at least 30 nucleotides, even if there is more than one mismatched base pair (Table 1); (b) The sequence with the highest percentage of mismatches in the conserved region is preferably positioned at the cleavage site of the DS primers (eg, hMPV 5'-585, hMPV 3'-698, and hMPV 3'-1007). ; (c) otherwise, mismatch nucleotides were placed at the 5'-terminal site, some of which could be replaced by universal bases such as deoxyinosine (eg, hMPV 3'-698 and hMPV 3'-1007) ; And (d) one or two nucleotides mismatched at the 3'-terminus can be replaced with degenerate or universal bases (eg, hMPV 3'-1007).

Genetic variation between hMPV isolates is indicated by underlined nucleotides.

DS primers specific for the hMPV F gene used in the Examples are as follows:

hMPV 5'-585 5'-AGCTTCAGTCAATTCAACAGAAIIIIICTAAATGTTG-3 '(39th sequence);

hMPV 3'-698 5'-AACATCAITTTTATITGTCCTGCAIIIIITGGCATGT-3 '(40th sequence); And

hMPV 3'-1007 5'-TTGAITGCTCAGCIACATTGATIIIIICWGCTGTGTC-3 '(SEQ ID NO. 41),

Wherein W is A or T and I is deoxyinosine.

Viral total RNA was extracted using RNAzol B method according to the manufacturer's protocol (RNAzol LS; Tel-Test, Inc.). Reverse transcription was performed to synthesize cDNA for 1.5 hours at 42 ° C. using viral RNA in a 20 μl reaction volume formulated as follows: 5 μl total RNA (approximately 100 ng), 4 μl 5 × buffer (Invitrogen, USA). ), 5 μl of dNTPs (5 mM each), 2 μl of 10 μM random hexadioxynucleotides, 0.5 μl of RNAase inhibitors (40 units / μl, Promega), and 1 μl of Moloney murine leukemia virus reverse transcriptase (200 units / μl, Promega).

2 μl primary cDNA chain (30 ng), 2 μl 10 × PCR buffer with 15 mM MgCl ₂ (Roche), 2 μl dNTP (2 mM each of dATP, dCTP, dGTP and dTTP), 5′hMPV-specific 1 μl of DS primer (hMPV 5'-585; 10 μM), 1 μl of 3'hMPV-specific DS primer (hMPV 3'-698 or hMPV 3'-1007; 10 μM), and Taq polymerase (5 unit / Target PCR amplification of the hMPV F gene in a final volume of 20 μl, including 0.5 μl of Roche; The tube containing the reaction mixture was placed in a preheated (94 ° C.) heat cycler; The sample was denatured at 94 ° C. for 5 minutes, followed by 30 minutes of 1 minute at 94 ° C., 1 minute at 60-65 ° C., and 1 minute at 72 ° C., followed by incubation at 72 ° C. for 7 minutes.

As can be seen in FIG. 10B, each pair of hMPV-specific DS primers (hMPV 5'-585 and hMPV 3'-698, and hMPV 5'-585 and hMPV 3'-1007), respectively, were expected at 150 bp and 459 Generate a single band corresponding to bp (lane 1 and lane 2). Subsequent sequence analysis of the product confirmed that the amplification product was a partial sequence of the hMPV fusion glycoprotein (F) gene. In contrast, the primers do not produce any product in negative control PCR without template (lane 4 and lane 5). Human beta-actin specific primer set was used as positive control (lane 3).

These results indicate that DS primers can be applied to detect hMPVs from patients with respiratory disease. Thus, for all situations encountered in the diagnostic art, it can be seen that the DS oligonucleotides of the present invention can be used as probes in primers or oligonucleotide chips of PCR amplification.

Example 7: DS oligo-fixed microarrays (Oligo-Immobilized Detection of Target Nucleotide Using Microarray )

Using a DNA synthesizer (Expedite 8900 Nucleic Acid Synthesis System, Applied Biosystems (ABI)), DS oligos complementary to one region of the target nucleic acid molecule are synthesized according to standard protocols. For microarrays, the synthesized DS oligos are fixed on glass slides. Then, 50-200 ng of DNA sample, 5 μl of 10 × PCR buffer (Promega), 15 mM MgCl ₂ A template-dependent extension reaction mixture comprising 5 μl, 5 μl of fluorescently-labeled dNTP (2 mM each of dATP, dCTP, dGTP and dTTP) and 0.5 μl of Taq polymerase (5 unit / μl; Promega) was added to the microarray. The microarray is then placed in a preheated (94 ° C.) heat cycler. The template-dependent extension reaction is carried out according to the following thermal cycle: 5 minutes at 94 ° C., followed by 1 minute at 94 ° C., 1-3 minutes at 50-65 ° C. and 1-4 minutes at 60-72 ° C. Repeat 15-50 times, then extend at 72 ° C. for 5 minutes. After the template-dependent extension reaction, the extended DS oligos are washed and a fluorescence image is obtained with a microarray scanner to detect the oligos, followed by the image analysis.

Example 8: DS Mononucleotide Polymorphisms ( SNPs ) with Oligonucleotides

DS oligos are synthesized using a DNA synthesizer (Expedite 8900 Nucleic Acid Synthesis System, Applied Biosystems (ABI)) according to standard protocols, where polymorphic bases (question sites) are located in the middle of the 3'-low Tm specificity site. The oligonucleotides synthesized for microarrays are fixed to glass slides. 50-200 ng of DNA sample, 5 μl of 10 × PCR buffer (Promega), 15 mM MgCl ₂ A template-dependent extension reaction mixture comprising 5 μl, 5 μl of fluorescently-labeled dNTP (2 mM each of dATP, dCTP, dGTP and dTTP) and 0.5 μl of Taq polymerase (5 unit / μl; Promega) was added to the microarray and The microarray is then placed in a preheated (94 ° C.) heat cycler. The template dependent extension reaction is carried out according to the following thermal cycles: after 5 minutes of denaturation at 94 ° C. for 1 minute at 94 ° C., 1-3 minutes at 50-65 ° C. and 1-4 minutes at 60-72 ° C. 15 Repeat 50 min and extend for 5 min at 72 ° C. Extended DS oligos are washed after the template-dependent extension reaction, oligos are detected through a fluorescence image obtained with a microarray scanner, and the image is analyzed.

As described above in detail specific parts of the present invention, modifications and variations within the spirit of the present invention will be apparent to those skilled in the art, the scope of the present invention is defined in the appended claims and equivalents thereof. Is determined by.

Claims (59)

A method of preparing a nucleic acid molecule by a template-dependent extension reaction using a bispecific oligonucleotide comprising the following steps: (a) annealing the bispecific oligonucleotide to a template nucleic acid molecule, wherein the bispecific oligonucleotide has a 5'-high T _m specificity site (5'-high T _m). specificity portion), 3'-low T _m specificity portion (5'-low T _m a specificity portion and a separation portion, three sites, wherein the 5'-high T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid to hybridize The site comprises at least two universal bases and the 3′-low T _m specific site has a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the template nucleic acid, and the annealing is a 3'-low T _m specific site. It is carried out under conditions in which annealing by itself does not occur; And (b) extending said bispecific oligonucleotide to produce a nucleic acid molecule complementary to said template nucleic acid molecule. Amplifying the target nucleic acid sequence using at least two cycles of primer annealing, primer extension, and denaturation, using a primer set comprising at least one bispecific oligonucleotide; The bispecific oligonucleotide comprises three sites, a 5'-high T _m specific site, a 3'-low T _m specific site, and a split site, wherein the 5'-high T _m specific site is one of the target nucleic acids that hybridizes. Having a hybridizing nucleotide sequence substantially complementary to position, wherein the cleavage site comprises at least two universal bases, and the 3′-low T _m specificity site is a hybridization nucleotide substantially complementary to one position of the target nucleic acid Has a sequence; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under conditions that the 5′-high T _m specific site and the 3′-low T _m specific site are annealed to the target nucleic acid; Annealing in the amplification reaction is a method for selectively amplifying a target nucleic acid sequence in a DNA or nucleic acid mixture, characterized in that the annealing by the 3'-low T _m specific site alone does not occur. At least one of the primer pairs comprises amplifying target nucleotide sequences by performing at least two cycles of primer annealing, primer extension, and denaturation using two or more primer pairs that are bispecific oligonucleotides, wherein the bispecific oligos The nucleotide comprises a 5′-high T _m specificity site, a 3′-low T _m specificity site and a cleavage site, three sites, wherein the 5′-high T _m specificity site is relative to one position of the nucleotide sequence to hybridize. Having a substantially complementary hybridizing nucleotide sequence, said cleavage site comprising at least two universal bases, and said 3'-low T _m specificity site comprises a hybridizing nucleotide sequence that is substantially complementary to one position of said target nucleotide sequence Have the 5'-high T _m special Cross portion T _m is higher than the 3'-low T _m specificity portion of the T _m, the cleavage site has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under conditions that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the target nucleotide sequence; Annealing in the amplification reaction is carried out under conditions in which no annealing by the 3′-low T _m specificity site alone occurs, so that two or more target nucleotide sequences are used simultaneously using two or more primer pairs. How to amplify. A method of sequencing a target nucleic acid molecule in a DNA or nucleic acid mixture using a bispecific oligonucleotide, comprising the following steps: (a) preparing a nucleic acid molecule complementary to the nucleic acid molecule to be sequenced by performing at least two cycles of primer annealing, primer extension, and denaturation using the bispecific oligonucleotide as a sequencing primer; The bispecific oligonucleotide comprises three sites, a 5'-high T _m specificity site, a 3'-low T _m specificity site, and a cleavage site, wherein the 5'-high T _m specificity site of the target nucleic acid molecule is hybridized. Having a hybridizing nucleotide sequence substantially complementary to one position, said cleavage site comprising at least two universal bases, and said 3'-low T _m specificity site is substantially complementary to one position of said target nucleic acid molecule Has a hybridizing nucleotide sequence; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the target nucleic acid molecule; Annealing in the preparation reaction is carried out under conditions in which annealing by the 3′-low T _m specific site alone does not occur; And (b) determining the nucleotide sequence of the prepared complementary nucleic acid molecule. A method for detecting nucleic acid molecules with genetic polymorphism by template-dependent extension of a bispecific oligonucleotide comprising the following steps: (a) annealing the bispecific oligonucleotide to the template nucleic acid molecule, wherein the bispecific oligonucleotide comprises a 5'-high T _m specific site, a 3'-low T _m specific site and a cleavage site, The 5′-high T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid to hybridize, the cleavage site comprises at least two universal bases, and the 3′-low T _m The specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the template nucleic acid; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a nonbase pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the template nucleic acid, and the annealing is a 3'-low T _m specific site. No annealing by itself occurs, and the 5'-high Tm specific site, the 3'-low Tm specific site or the 5'-high Tm specific site and the 3'-low Tm specific site with respect to the target position. Annealing occurs under conditions that occur when one or more mismatched bases are present; (b) extending said bispecific oligonucleotide to produce said nucleic acid molecule complementary to said template; And (c) detecting whether the template-dependent extension reaction of said bispecific oligonucleotide occurs. A method of detecting a target nucleotide sequence in a nucleic acid sample by template-dependent extension of a bispecific oligonucleotide comprising the following steps: (a) extending a bispecific oligonucleotide as a probe immobilized on a substrate, comprising at least one cycle of hybridization, template-dependent extension and denaturation, wherein said hybridization is effected by contacting the bispecific oligonucleotide with a nucleic acid sample Wherein the bispecific oligonucleotide has a 5′-high T _m specificity site (5′-high T _m specificity portion), 3'-low T _m specificity portion (5'-low T _m a specificity portion and a separation portion, three sites, wherein the 5'-high T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the target nucleotide sequence to hybridize, and The cleavage site comprises at least two universal bases and the 3′-low T _m specificity site has a hybridizing nucleotide sequence that is substantially complementary to one position of the target nucleic acid; The 5'-high T _m specificity portion of the T _m is higher than T _m of the 3'-low T _m specificity portion, the dividing portion has the lowest T _m among the three portions; The cleavage site forms a non-base pair bubble structure under the condition that the 5'-high T _m specific site and the 3'-low T _m specific site are annealed to the target nucleotide sequence, and the annealing in the amplification reaction is 3'-low. Carried out under conditions in which annealing by the T _m specific site alone does not occur; And (b) analyzing whether a template-dependent extension reaction has occurred. The method according to any one of claims 1 to 6, wherein the universal base in the cleavage site 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 '-Dioxy-beta-D-ribofuranosyl) -3-nitropyrrole, deoxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindole , Dioxy 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-nitro Indole, morpholino-nebulin, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholi 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 combinations of said bases. 8. The method of claim 7, wherein the universal base is deoxyinosine, 1- (2'-deoxy beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole. 9. The method of claim 8, wherein said universal base is deoxyinosine. 7. The method of any one of claims 1 to 6, wherein the cleavage site comprises a sequence of nucleotides having a universal base. The method of any one of claims 1 to 6, wherein the 5'-high T _m specific site is longer than the 3'-low T _m specific site. 7. The method of any one of claims 1 to 6, wherein the 3'-low T _m specificity site has a hybridizing nucleotide sequence that is perfectly complementary to the position of the template nucleic acid to hybridize. The method of any one of claims 1 to 6, wherein the 5'-high T _m specificity site has a length of 15 to 40 nucleotides. The method of claim 13, wherein the 5′-high T _m specificity site has a length of 15 to 25 nucleotides. 7. The method of claim 1, wherein the cleavage site has at least 3 nucleotides in length. 7. The method of any one of claims 1 to 6, wherein said cleavage site has a length of 3 to 10 nucleotides. 7. The method of claim 1, wherein the 3′-low T _m specificity site has a length of 3 to 15 nucleotides. 7. The method of claim 1, wherein the 5′-high T _m specificity site is 15-25 nucleotides in length, the cleavage site is 3-10 nucleotides in length and the 3′-low T _m specificity site is And 3 to 15 nucleotides in length. Any one of claims 1 to A method according to any one of claims 6 or 7, characterized in that the 5'-high T _m specificity portion of the T _m is 40 ℃ -80 ℃. Claim 1 to 6 according to any one of items, characterized in that it said the 3'-low T _m specificity portion has a T _m 10 ℃ -40 ℃. The method of claim 1, wherein the T _m of the cleavage site is 3 ° C.-15 ° C. 8. The method of claim 1, wherein the annealing is carried out at a temperature in the range of 45 ° C.-68 ° C. 8. 6. The method of claim 1 or 5, wherein the preparation of the target nucleic acid sequence is achieved by repeating a process of template-dependent extension reaction including the bispecific oligonucleotide annealing, extension and denaturation. The method of claim 2 or 3, wherein the amplification reaction is carried out according to the polymerase chain reaction. The method of claim 4, wherein the target nucleic acid molecule to be sequenced is contained within genome DNA or cDNA populations. 6. The method of claim 5, wherein the nucleic acid molecule having genetic diversity is a nucleic acid of a virus having genetic diversity. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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