KR100812259B1 - Method for Amplifying Unknown Sequence Adjacent to Known Sequence - Google Patents

Abstract

The present invention includes a step (a) of primary amplification of an unknown nucleotide sequence using a DNA working annealing regulatory primer (DW-ACP) and a primary target-specific primer, wherein the step (a) a) relates to a method for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence comprising the following steps: (a-1) hybridizing to the unknown nucleotide sequence At least one cycle of primer annealing, primer extension, and denaturation using a primary degenerate DW-ACP comprising a degenerate random nucleotide sequence and a hybridizing nucleotide sequence substantially complementary to a location in the unknown nucleotide sequence A first-stage amplifica of the unknown nucleotide sequence at a first annealing temperature tion); And (a-2) performing a second-stage amplification at a second annealing temperature that prevents the first degenerate DW-ACP from operating as a primer.

DNA Walking, Primer, ACP, Degenerate, Amplification

Description

Method for Amplifying Unknown DNA Sequence Adjacent to Known Sequence

The present invention relates to a method for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, and more particularly, to amplify an unknown nucleotide sequence using a novel DNA working annealing control primer and its application. It is about.

Polymerase chain reaction (PCR) is the most effective way to selectively amplify specific DNA fragments. In the PCR process, oligonucleotides complementary to the 5 'and 3' sequences adjacent to the target nucleic acid are used as "primers," which play an important role.

When PCR is applied to isolate and analyze specific DNA sites, information is required about DNA sequences adjacent to the site of interest. This requirement only allows amplification of the site of the known DNA sequence. In the absence of necessary sequence information, PCR amplification of target DNA fragments in complex DNA populations can result in amplification of non-target DNA.

Many PCR-based methods have been developed to separate unknown DNA sequences adjacent to known sequences. These methods include inverse PCR (Triglia et al., 1988), platehandle PCR (Shyamala et al., 1989; Jones and Winistorfer, 1997), vectorlet PCR (Arnold et al., 1991), anchored PCR (Roux). et al., 1990), AP-PCR (Dominguez et al., 1994; Trueba and Johnson, 1996), capture PCR (Lagerstrom et al., 1991) and adapter- or cassette-linked PCR (Iwahana et al., 1994 Riley et al., 1990; Siebert et al., 1995; Willems, 1998; Kilstrup and Kristiansen, 2000).

However, the above methods require the cleavage of DNA with restriction enzymes, ligation of the cleaved DNA with a linker or double chain, partial double or single chain oligonucleotide cassette, followed by purification and / or subcloning of the product prior to sequencing. It has the following limitations. The need for these different procedures reduces the convenience and efficiency of the above methods. In addition, in the above methods, common problems are found, such as the formation of high background and non-specific products caused by non-specific binding of vectors, adapters, cassettes or tail primers.

Therefore, a biotin / streptavidin system that captures fragments for biotinylation prior to conducting nested PCR has been proposed as a novel methodology (Rosenthal and Jones, 1990; Mishra et al. 2002). This method represents an improvement in reducing noise and allowing amplification of the site adjacent to the known sequence, but requires a complex immobilization step and is expensive.

Throughout this specification, numerous patent documents and articles are referenced and their citations are shown in parentheses. The disclosures of patent documents and articles are incorporated by reference herein in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

In order to overcome the shortcomings of the conventional techniques described above, the present inventors have tried to develop a method that can basically completely eliminate high background problems in amplifying unknown sequences, and as a result, it is more convenient with high reliability. We have developed a novel method of amplifying an unknown sequence adjacent to a known sequence that can amplify the unknown sequence in a manner.

Accordingly, it is an object of the present invention to provide a method for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence.

Another object of the present invention is to provide a DNA working annealing regulatory primer for amplifying an unknown nucleotide sequence.

Another object of the present invention is to provide a kit for amplifying an unknown nucleotide sequence.

Another object of the present invention is to provide the use of the above method for a process comprising the process of nucleic acid amplification of an unknown nucleotide sequence adjacent to a known nucleotide sequence.

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

According to an aspect of the present invention, the present invention provides a method for performing a primary amplification of an unknown nucleotide sequence using a DNA working annealing regulatory primer (DW-ACP) and a primary target-specific primer (a Wherein step (a) provides a method for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence comprising the following process: (a-1) A primer using a first degenerate DW-ACP comprising a degenerate random nucleotide sequence that hybridizes to the unknown nucleotide sequence and a hybridizing nucleotide sequence that is substantially complementary to a position of the unknown nucleotide sequence Primary annealing, including at least one cycle of annealing, primer extension, and denaturation, wherein a first degenerate DW-ACP extension is produced Performing a first-stage amplification of the unknown nucleotide sequence at a temperature, wherein the primary annealing temperature causes the primary degenerate DW-ACP to act as a primer (a- 2) performing a second-stage amplification at a second annealing temperature that prevents the first degenerate DW-ACP from acting as a primer, comprising: (a -2-1) the primary degenerate DW-ACP extension using the primary target-specific primer hybridizing to a target-specific nucleotide sequence that is substantially complementary to a position on the unknown nucleotide sequence Amplifying the target-specific primer extension product (a-2-2) complementary to the first degenerate DW-ACP sequence of the target-specific primer extension product. Nuckle Amplifying the target-specific primer extension using a second DW-ACP that hybridizes to an ideide sequence, whereby a second DW-ACP extension is produced, and (a- 2-3) amplifying the secondary DW-ACP extension using the secondary DW-ACP and the primary target-specific primer, wherein the step is free of degenerate random nucleotide sequences. A primary amplification product is produced.

The present invention provides a unique method for selectively amplifying unknown DNA sequences adjacent to a portion of a known sequence from a DNA or nucleic acid mixture using novel DNA working annealing control primers (hereinafter referred to as “DW-ACP”). It is about.

In order to overcome the problems of conventional DNA (or genome) working methods such as complex and numerous steps and the inherent background problems of PCR-based methods, the annealing control primers disclosed in WO 03/050305 developed by the inventors An annealing control primer (ACP) system was modified and subjected to selective amplification of unknown sequences adjacent to a portion of the known sequence. Since the ACP system can greatly improve the specificity of the amplification reaction, using ACP can not only fundamentally inhibit the non-specific priming of primers in the amplification process but also simplify the amplification process in the present invention. have.

According to a preferred embodiment of the invention, said first degenerate DW-ACP is represented by the following general formula (I):

5'-X _p -Y _q -Z _r -Q _s -3 '(I)

Wherein X _p represents a 5′-terminal site having a pre-selected nucleotide sequence, and Y _q represents a regulatory site comprising at least two universal bases or non-differential base analogs, wherein Z _r Represents a degenerate random sequence site having a degenerate random nucleotide sequence, Q _s represents a 3′-terminal site having a hybridizing nucleotide sequence substantially complementary to one position of the unknown nucleotide sequence, p, q, r and s is the number of nucleotides; X, Y, Z and Q are deoxyribonucleotides or ribonucleotides.

The primary degenerate DW-ACP of the present invention was developed to amplify an unknown nucleotide sequence adjacent to a known nucleotide sequence, utilizing the principles of annealing regulatory primers disclosed in WO 03/050305 developed by the inventors. And the teachings of this patent document are incorporated herein by reference.

The principle of primary degenerate DW-ACP is the composition of oligonucleotide primers with distinct 3'- and 5'-terminal sites separated by at least two universal or non-specific base analogs, and oligonucleotide primers. In effect of the regulator site on the 3'- and 5'-terminal sites. The presence of a regulator site between the 3′- and 5′-terminal sites of the primary degenerate DW-ACP is a major factor leading to an improvement in primer annealing specificity.

The term “nucleic acid” or “nucleotide” is a deoxyribonucleotide or ribonucleotide polymer in single or double chain form and includes known analogs of natural nucleotides, unless specifically noted otherwise. Thus, the primary degenerate DW-ACP of the present invention can be used for nucleic acid amplification using single or double chain gDNA, cDNA or mRNA templates. The term "portion" as used in connection with a primer of the present invention refers to a nucleotide sequence separated by an interventional site, such as a regulator site. The term “3-terminal site” or “5-terminal site” means a nucleotide sequence at the 3′-end or 5′-end of the primers of the invention, isolated at the regulator site.

As used herein, the term “primer” refers to a synthetic or natural oligonucleotide. The primer serves as a starting point for the synthesis under conditions in which the synthesis of the primer extension product complementary to the nucleic acid chain (template) is induced, i.e., the presence of a polymerizer such as nucleotides and DNA polymerase, and conditions of suitable temperature and pH. For maximum efficiency of amplification, the primer is preferably single chain. Preferably, the primer is an oligodioxyribonucleotide. Primers of the invention may include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides.

The term “substantially complementary” as used in connection with a primer means that it is sufficiently complementary that the primer selectively hybridizes to the template nucleic acid sequence under certain annealing conditions, and the annealed primer is extended by a polymerase. To generate the complement of the nucleotide sequence, thus the term has a meaning different from that of the term “perfectly complementary” or related thereto.

It is known that nucleotides of uncertain positions in degenerate primers are replaced by universal or non-specific base analogs, which are known as deoxyinosine (Ohtsuka et al, 1985; Sakanari et al., 1989), 1- (2'-deoxy-beta-D-ribofuranosyl) -3-nitropyrrole (Nichols et al., 1994) and 5-nitroindole (Loakes and Brown, 1994), the bases It is used to solve problems related to the design of degenerate primers because they are base-paired nonspecifically with four conventional bases. However, universal or non-differential base analogs, such as deoxyinosine, 1- (2'-deoxy-betaD-ribofuranosyl) -3-nitropyrrole and 5-nitroindole, have unknown nucleotide sequences. There is no report that it can serve as a regulator to distinguish each functional site in the primer according to the annealing temperature in the primer for amplifying the DNA working primer.

The term “universal base or non-discriminatory base analog” is capable of forming base pairs with each of the natural DNA / RNA bases without distinguishing them from the natural DNA / RNA bases. Means that.

According to a preferred embodiment of the invention, the universal base or non- discriminatory base analog is dioxyinosine, 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, deoxy nebulin, 2'-F nebulin, 2'-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, Morpholino-nebulin, morpholino-inosine, morpholino-4-nitrobenzimidazole, mother Porino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebulin, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphorami Date-3-nitropyrrole, 2'-0-methoxyethylinosine, 2'-0-methoxyethyl nebulin, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxy Ethyl 4-nitro-benzimidazole, 2'-0-methoxyethyl 3-nitropyrrole and combinations of these bases, including, but not limited to. More preferably, the universal base or non- discriminatory base analog is dioxyinosine, inosine, 1- (2'-dioxy-beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole And most preferably, deoxyinosine.

The presence of polydioxynucleotides with universal bases such as deoxyinosine in the primer results in low annealing temperatures due to the weak hydrogen bond interactions in the base pairs. Extending this theory, the present inventors form a portion having a low melting temperature in the presence of polydioxynucleotides having universal bases between the degenerate sequence region of the primer and the 3'- and 5'-terminal sites. This portion forms a boundary at each of the degenerate sequence site and the 3'- and 5'-end sites, which will facilitate annealing of the degenerate sequence site and the 3'-end site to the target sequence at a specific temperature. Deduced the fact that This theory provides the basis for the primary degenerate DW-ACP of the present invention.

The regulator site in the primary degenerate DW-ACP may regulate the annealing site of the primer (ie, the degenerate random sequence and the 3′-terminal site) depending on the annealing temperature. The regulator site not only inhibits the 5′-terminal site sequence from annealing the template, but also acts to limit the annealing site of the primer to the degenerate sequence site and the 3′-terminal site at the primary annealing temperature. The modulator site thus greatly improves annealing for the template of the degenerate sequence site and the 3′-terminal site of the primary degenerate DW-ACP.

According to a preferred embodiment of the invention, the regulator region of the primary DW-ACP comprises at least three universal bases or non-differential base analogs between the 5'-terminal region and the degenerate sequence region, more Preferably at least four universal bases or non- discriminatory base analogs. Advantageously, the length of the universal base residue between the 5′-terminal site and the degenerate sequence site of the primary DW-ACP is at most 10 residues. According to a preferred embodiment of the invention, the regulator region of the primary DW-ACP comprises 2-10 universal bases or non- discriminatory base analogs. Most preferably, the universal base between the 5′-terminal site and the degenerate sequence site of the primary degenerate DW-ACP is about 3-5 residues in length. Universal bases or non- discriminatory base analogs may be present in a continuous or intermittent, preferably in a continuous manner.

The 5′-terminal portion of the primary degenerate DW-ACP partially contributes to the annealing specificity. Importantly, the 5'-terminal site acts as the priming site either alone or in combination with other sites in subsequent amplification reactions following the first round of amplification reactions. According to a preferred embodiment, the pre-selected nucleotide sequence of the 5'-terminal region is substantially not complementary to any position on the template nucleic acid.

In general, the 5′-terminal portion of the primary degenerate DW-ACP comprises at least 10 nucleotides. Preferably, the 5′-terminal region sequence is at most 60 nucleotides in length. More preferably, the 5'-terminal region sequence is 6-50 nucleotides, most preferably 18-25 nucleotides in length. If longer sequences are used at the 5′-terminal site, the efficiency of the primary degenerate DW-ACP can be reduced, and if shorter sequences are used, the annealing efficiency can be reduced under high stringency conditions.

In one embodiment of the invention, the pre-selection nucleotide sequence of the 5'-terminal region may comprise universal primer sequences such as T3 promoter sequence, T7 promoter sequence, SP6 promoter sequence and M13 forward or reverse universal sequence. .

According to one embodiment of the present invention, the 5′-terminal portion of the primary degenerate DW-ACP may be modified within a range that does not impair the advantages of DW-ACP, that is, improvement of annealing specificity. For example, the 5′-terminal portion may comprise the recognition sequence (s) of the restriction enzyme (s), which sequence facilitates cloning the amplification product into a suitable vector. In addition, the 5′-terminal portion may comprise at least one nucleotide having a label for detection or separation of the amplification product. Suitable labels include fluorophores, chromophores, chemilumines, magnetic particles, radioisotopes, mass labels, electron dense particles, enzymes, cofactors, substrates for enzymes, and antibodies, streptavidin, biotin, digoxigenin and chelating Hapten having a specific binding partner, such as a group, including but not limited to. The 5′-terminal portion may also comprise a bacteriophage RNA polymerase promoter portion.

The degenerate random sequence region of the primary degenerate DW-ACP is located between the regulator and the 5′-terminal region. The term “degenerate random sequence” site refers to a nucleotide sequence in which each nucleotide is occupied by any of four deoxyribonucleotides, ie dATP, dTTP, dCTP and dGTP. The degenerate random sequence site thus provides a pool of primers having various nucleotide sequences, at least one of which is expected to be annealed at any location on the unknown target sequence of the template. For example, if a primary degenerate DW-ACP comprising three degenerate nucleotides is synthesized at a degenerate random sequence site, 64 oligonucleotides may be generated. Thus, the use of degenerate random sequence sites further increases the possibility of primary degenerate DW-ACPs hybridizing to unspecified target nucleic acids. When the target core sequence of the primary degenerate DW-ACP comprising the degenerate sequence and the 3'-terminal region sequence hybridizes to one position of the template, the degenerate region and the 3 'of the primary degenerate DW-ACP Several complementary binding positions of the terminal region sequences are present in the unknown target sequence of the template. Interestingly, the inventors believe that the primary degenerate DW-ACP binds to one target-binding position at the closest distance from the target-specific primer sequence of the known sequence, thereby generally producing one main target product. The facts were observed. Primers that hybridize to other binding sites that are located at a location further apart from the target-specific primer sequence do not produce products well because the primers hybridized to the binding site closest to the target-specific primer sequence are extended. This is because this may interfere with the extension of primers hybridized to other binding sites.

Depending on various considerations such as the length of the 3′-terminal site, the amplification reaction yield and the length of the nucleotide to be amplified, the length of the degenerate random sequence site of the primary degenerate DW-ACP can be determined. For example, as the length of the degenerate sequence region increases, amplification yields decrease in the present invention. In general, the length of the degenerate sequence region is 1-5, preferably 2-5, more preferably 2-4 and most preferably 3.

The 3′-terminal portion of the primary degenerate DW-ACP has a nucleotide sequence that is substantially complementary to a location on an unknown nucleotide sequence. The 3'-terminal portion of the primary degenerate DW-ACP is one or more misses to one position of the unknown nucleotide sequence within the range in which the primary degenerate DW-ACP can act as a primer. It is natural to have a match. Most preferably, the 3′-terminal portion of the primary degenerate DW-ACP has a nucleotide sequence that is completely complementary to one location of the unknown nucleotide sequence, ie there is no mismatched sequence.

The 3′-terminal portion of the primary degenerate DW-ACP has a nucleotide sequence that hybridizes to a position in an unknown sequence, ie an “arbitrary” nucleotide sequence. The term “aviary nucleotide sequence” means a nucleotide sequence that is selected without information about the unknown nucleic acid sequence to be amplified.

In general, the 3′-terminal portion of the primary degenerate DW-ACP is at least 3 nucleotides in length. It is important that the annealing site (ie, the degenerate sequence and the 3′-terminal site) of the primary degenerate DW-ACP is at least 6 nucleotides in length, which is the minimum required for the primer to anneal with specificity. Judging by the length requirement. In practice, primer annealing with specificity can be secured when the annealing site is at least 8 nucleotides. Preferably, the length of the 3′-terminal portion is 3-20 nucleotides, more preferably 3-10 nucleotides and most preferably 4-6 nucleotides.

The 3′-terminal portion of the primary degenerate DW-ACP is prepared to have a particular abiary nucleotide sequence. The particular abiary nucleotide sequence may comprise a cossack sequence of mRNA comprising a translational initiation codon ATG such as 5'-ANNATGN-3 ', wherein N is any one of four deoxynucleotides (McBratney and Sarnow). , 1996). In addition, the particular abiary nucleotide sequence may comprise a standard polyadenylation signal sequence AATAAA (Juretic and Theus, 1991). Optionally, the nucleotide at the 5'-terminus of the 3'-terminus portion of the primary degenerate DW-ACP can be varied to have any one of the four deoxynucleotides, thereby Four primary degenerate DW-ACPs are obtained in terms of the nucleotide sequence.

The length of the 3′-terminal portion of the primary degenerate DW-ACP can be determined based on various considerations such as the length of the nucleotide to be amplified, the length of the degenerate sequence region and the amplification yield. For example, where the 3′-terminal site has four abitary nucleotides, theoretically 4-base sequence combinations may exist once every 256 bp in the template. Thus, the length of the nucleotide to be amplified is at least 256 bp.

The total length of the primary degenerate DW-ACP is preferably 20-80 nucleotides, more preferably 28-50 nucleotides, and most preferably 30-40 nucleotides.

In the methods of the invention, a primary target-specific primer is used that is substantially complementary to a position on the known nucleotide sequence of the template. The term “substantially complementary” used while referring to a primary target-specific primer has the same meaning as that used for the primary degenerate DW-ACP.

In the present invention, primary amplification is carried out according to a two-step amplification process carried out at different annealing temperatures in order to maximize the advantages of the DW-ACP system.

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

If mRNA is used as the starting material, a reverse transcription step is required before amplification, and details of the reverse transcription step can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001). And Noonan, KF et al., Nucleic Acids Res. 16: 10366 (1988). In the reverse transcription step, oligonucleotide dT primers are used that hybridize to the poly A tail of the mRNA. The reverse transcription step can be accomplished by reverse transcriptase.

The method of the present invention does not require that the molecule to be amplified has a particular sequence or length. In particular, molecules that can be amplified include natural prokaryotic nucleic acids, eukaryotic cells (eg, protozoan and parasitic animals, 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 acids and non-loid nucleic acids. In addition, the nucleotide sequence includes nucleic acid molecules that can be chemically synthesized or can be synthesized. Thus, the nucleotide sequence is either found in nature or not found.

The primary degenerate DW-ACP used in the present invention hybridizes or anneals at one position on an unknown nucleotide sequence, and eventually forms a double-chain structure. Conditions for nucleic acid hybridization suitable for forming such double chain structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985). The sequence of the annealing site (ie, the degenerate sequence and the 3′-terminal site) of the primary degenerate DW-ACP need not exhibit complete complementarity, but is substantially complementary enough to form a stable double chain structure. Just have a sequence. Thus, departures from complete complementarity are permitted, provided that the hybridization for the formation of the double-chain structure is not completely excluded. Hybridization of a primary degenerate DW-ACP at a location on an unknown sequence is a prerequisite for template-dependent polymerization by polymerase. Factors affecting the formation of base pairs of primary degenerate DW-ACPs in complementary nucleic acids (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)) affect priming efficiency. The nucleotide composition of the primary degenerate DW-ACP affects the optimum annealing temperature and thus also the priming efficiency.

Various DNA polymerases can be used in the amplification step of the present invention and include “Clenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which 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.

All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions.

The two stage amplification process of the present invention is only separated in time. The first stage amplification is followed by the second stage amplification. Thus, the two-stage amplification can be carried out in one reaction system comprising all kinds of primers, primary DW-ACP, secondary DW-ACP, and primary target-specific primers.

Annealing or hybridization in the present invention is carried out under stringent conditions that allow specific binding between the nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary with ambient environmental variables. In the present invention, the two-step amplification process is carried out at different conditions, in particular at different annealing temperatures. Preferably, the first stage amplification is carried out at low stringency conditions, in particular at low annealing temperatures. More preferably, the primary annealing temperature is about 35-50 ° C., most preferably 40-48 ° C. At the primary annealing temperature, the annealing site of the primary DW-ACP is defined as the degenerate sequence and the 3′-terminal site, which improves the annealing specificity.

According to the present invention, the first stage amplification process under low stringency conditions, in order to improve the specificity of primer annealing in the first stage amplification, performs at least one cycle of annealing, extension and denaturation, and then the second stage amplification The process proceeds more effectively under high stringency conditions. Most preferably, the first stage amplification process is performed one cycle. One cycle of the first stage amplification process is important because it inhibits the formation of non-specific amplification products with primary degenerate DW-ACP alone. During one cycle of first amplification (first stage amplification), the 3′-terminal portion of the first degenerate DW-ACP binds to the unknown target positions of the template under low stringency conditions. However, in other cycles of primary amplification (second stage amplification), the 3'-terminal portion of the primary degenerate DW-ACP is highly stringent, except for the nucleotide sequence complementary to the primary DW-ACP. It does not act as a primer that binds to a template or binds to an extended primer sequence under conditions. As a result, the primary degenerate DW-ACP alone cannot produce any product during the primary amplification except for the primary degenerate DW-ACP extension product (see FIG. 2).

The second stage amplification process is preferably carried out at high salt conditions, in particular at high annealing temperatures. Advantageously, the secondary annealing temperature is about 50-72 ° C., more preferably 50-65 ° C. and most preferably 52-65 ° C. At high annealing temperatures, such as secondary annealing temperatures, the primary degenerate DW-ACP no longer acts as a primer; Instead, the secondary DW-ACP and primary target-specific primers act as primers with high specificity.

The second stage amplification process under high stringency conditions is carried out for at least one cycle, preferably at least five cycles, using a combination of the second DW-ACP and the first target-specific primer, whereby the first order An unknown target-specific product is created without degenerate random nucleotide sequences derived from degenerate DW-ACP. More preferably, the second stage amplification is carried out for 10-40 cycles, even more preferably 10-30 cycles, and most preferably 10-20 cycles.

High stringency and low stringency conditions can be readily determined from standards known in the art. The term “cycle” refers to the process of producing one copy of a target nucleic acid. The cycle includes denaturation, annealing and extension processes.

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

According to a preferred embodiment of the invention, the secondary degenerate DW-ACP is represented by the following general formula II:

5'-X ' _p -S _u -Y' _v -Z ' _w -3' (II)

In the general formula, X ' _p represents a 5'-terminal portion having a nucleotide sequence corresponding to the 5'-terminal portion of the primary degenerate DW-ACP, S _u of step (a-2-1) target-specific primers represents the extension product of the first degeneracy DW-ACP regulator portion of the secondary annealing region comprising a nucleotide sequence that hybridizes to the other end portion for, Y _'v is the first degeneracy DW- ratio than the complementary nucleotide sequence to the ACP - the target sequence X 'and _p S _u A modulator site comprising at least two universal bases or non-distinguishing base analogs that interfere with the site being annealed, Z ' _w corresponds to the 3'-terminal site of the primary degenerate DW-ACP A 3'-terminal site having a nucleotide sequence, wherein p, u, v and w are the number of nucleotides; X ', S, Y', and Z 'are deoxyribonucleotides or ribonucleotides.

The term “corresponding to”, used to refer to two related sequences, is a sequence that is completely identical or partially identical to one another such that one nucleotide sequence hybridizes to a nucleotide sequence that can hybridize with another nucleotide sequence of another comparison. Is a term used to express.

The secondary DW-ACP of Formula II has a nucleotide sequence corresponding to the sequence of the primary degenerate DW-ACP. Primers of formula II exhibit higher annealing specificity.

The 5′-terminal portion of the secondary DW-ACP has a nucleotide sequence corresponding to the 5′-terminal portion of the primary DW-ACP. That is, the nucleotide sequence of the 5′-terminal portion of the secondary DW-ACP is completely or partially identical to the sequence of the 5′-terminal portion of the primary DW-ACP. Completely exclude hybridization that results in the formation of a double-chain structure between the 5'-terminal portion of the secondary DW-ACP and the nucleotide sequence having an opposite sense with respect to the 5'-terminal portion of the primary DW-ACP To the extent it does not, deviations from perfect identity are allowed.

The secondary annealing site (S _u ) in the second DW-ACP of Formula II is very unique, which contributes to some degree in completely eliminating high background problems resulting from nonspecific binding of primers to nonspecific sites. to be. The secondary annealing site comprises a nucleotide sequence that hybridizes to the site opposite to the regulator site of the primary degenerate DW-ACP. The strategy for this design of the auxiliary annealing site is to use DNA polymerase (eg, Taq polymerase) to recognize universal bases and direct the insertion of native NMPs. The way DNA polymerases recognize universal bases is described by Geoffrey C. Hoops, et al. Nucleic Acids Research, 25 (24): 4866-4871 (1997), which is incorporated herein by reference, for example 8-hydroxyguanine, 2-hydroxyadenine, 6- O-methylguanine and xanthine direct the insertion of (C and A), (T and A), (T and C) and (T and C), respectively. Geoffrey C. Hoops, et al. Bases comprising nitropyrrole and inosine, most preferably, indicate direct insertion of dAMP and dCMP, respectively.

Thus, when the regulator site of the primary degenerate DW-ACP contains at least two deoxyinosine or inosine residues, the deoxycytidine nucleotide is most preferably by the Taq polymerase on the opposite site of the regulator site. Since inserted, the secondary annealing site should contain at least two deoxyguanosine nucleotides.

In the secondary DW-ACP, Y ' _v , the modulator site is X' _p and S _u in the non-target sequence except for the nucleotide sequence complementary to the primary degenerate DW-ACP. Suppresses annealing of the site. In addition, the modulator site provides an additional annealing site through non-differentiating binding of universal base or non-differential base analogs, which means that the secondary DW-ACP is the primary degenerate DW in the resulting product. Allow selective annealing to sequences complementary to the ACP sequence.

The regulator site of Formula II hybridizes to the site opposite to the degenerate sequence site of the primary degenerate DW-ACP. Universal bases or non-differential base analogs suitable for the regulator site can be any base that loses its distinction when participating in DNA replication. Preferably, the universal base or non- discriminatory base analog is dioxyinosine, inosine, 7-diaza-2'-dioxyinosine, 2-aza-2'-dioxyinosine, 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-nitroindole, deoxy 4-nitrobenz Imidazole, 4-nitrobenzimidazole, dioxy 4-aminobenzimidazole, 4-aminobenzimidazole, dioxy nebulin, 2'-F nebulin, 2'-F 4-nitrobenzimidazole , PNA-5-introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebula Lean, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosph Lamidate-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 these bases. More preferably, the universal base or non- discriminatory base analog is dioxyinosine, inosine, 1- (2'-dioxy-beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole And most preferably, deoxyinosine.

The length of the regulator site of Formula II is mainly determined by the length of the degenerate sequence site of the primary degenerate DW-ACP. In addition, when the nucleotide at the 5'-end of the 3'-end site of the primary degenerate DW-ACP is designed to have a specific nucleotide, the regulator site is lengthened by one nucleotide. The universal base at the regulator site of formula II is preferably present in a continuous manner.

The 3′-terminal portion of the secondary DW-ACP has a nucleotide sequence corresponding to the 3′-terminal portion of the primary degenerate DW-ACP. The nucleotide sequence of the 3′-terminal portion of the secondary DW-ACP is completely identical or partially identical to the sequence of the 3′-terminal portion of the primary degenerate DW-ACP. Hybridization allows formation of a double-chain structure between the 3'-terminal portion of the secondary DW-ACP and the nucleotide sequence having an opposite sense to the 3'-terminal portion of the primary degenerate DW-ACP. To the extent that not entirely excluded, departures from perfect identity are permitted. Most preferably, the nucleotide sequence of the 3'-terminal portion of the secondary DW-ACP is completely identical to the 3'-terminal portion of the primary degenerate DW-ACP, which is the 3'-terminus of the primer in the template. This is because complete site agreement is required for successful amplification.

According to the second stage of amplification, the primary target specific primers anneal to their complementary sequences on known nucleotide sequences to produce a target-specific primer extension product. If the regulator site of the primary degenerate DW-ACP comprises a universal base or a non-differential base analogue, its opposite strand in the target-specific primer extension product is DNA polymerized as described above. Nucleotides preferred by the enzyme. For example, if the regulator site of the primary degenerate DW-ACP comprises at least two deoxyinosine or inosine residues, its opposite chain in the target-specific primer extension product may have at least two deoxy Tydine nucleotides are inserted.

After the target-specific primer extension product is generated, the primary amplification product is generated using the second DW-ACP and the primary target-specific primer. During the second stage amplification process, all sites of the secondary DW-ACP are annealed only to the nucleotide sequence complementary to the primary DW-ACP sequence located at the 3'-end of the amplification product, but not to other positions due to the modulator. can not do it. Thus, secondary DW-ACP alone cannot produce non-specific products (see FIG. 2).

The final primary amplification product without the degenerate random sequence derived from the primary DW-ACP is used as a template in the secondary amplification process. When the second amplification is carried out following the first amplification, the second amplification process is performed with higher specificity and reliability since the first amplification product does not have a degenerate random sequence. It is described.

According to a preferred embodiment of the present invention, the method of the present invention comprises a nucleotide sequence that hybridizes with a counter-sense nucleotide sequence for the second DW-ACP sequence present at the 3'-end of the primary amplification product. A third DW-ACP comprising at the 3'-terminal site and a nested target-specific primer for amplifying the primary target-specific primer of step (a) or the internal site of the primary amplification product (C) subjecting the secondary amplification to a third annealing temperature, comprising at least one cycle of primer annealing, primer extension and denaturation employed.

Preferably, the second amplification reaction follows a nested amplification procedure known in the art, for selectively amplifying the unknown target product from the first amplification product. Preferably, the second amplification reaction is carried out at high salt conditions, in particular at high annealing temperatures. Advantageously, the high annealing temperature is about 50-72 ° C., more preferably 50-70 ° C. and most preferably 55-68 ° C. At high annealing temperatures, all but part of the tertiary DW-ACP are involved in the annealing. Thus, the third DW-ACP may be selectively annealed only to the nucleotide sequence complementary to the second DW-ACP. The second amplification reaction under high salt conditions is carried out for at least one cycle, preferably at least five cycles, whereby the first amplification product is amplified. In a more preferred embodiment, the second amplification reaction is carried out for 10-40 cycles.

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

According to a preferred embodiment of the invention, the third DW-ACP is represented by the following general formula III:

5'-X ” _p -Y” _q -C _c -3 '(III)

Wherein X ″ _p represents a 5′-terminal portion having a nucleotide sequence corresponding to all or a portion of the 5′-terminal portion of the secondary DW-ACP, and Y ″ _q represents a second of Formula II At least two of the secondary annealing site, a portion of the 5'-terminal site, a portion of the secondary annealing site and a portion of the 5'-terminal site, or a portion of the secondary annealing site and the regulator site of the primary DW-ACP; Annealing the X ″ _p site to a non-target sequence of a primary amplification product, excluding a nucleotide sequence complementary to a second DW-ACP, indicating a regulator site comprising a universal base or a non-specific base analog act to interfere with, and, C _c is the second DW-ACP of the 3'-end region and the regulator portion opposite with respect to all or a portion of the sequence - 3 ' end of the sense nucleotide sequence has a nucleotide sequence which hybridizes with the It denotes a portion, p, q, and c is the number of nucleotides, X ", Y", and C is a deoxyribonucleotide or a ribonucleotide.

In the tertiary DW-ACP, X ″ _p has a nucleotide sequence corresponding to all of the 5′-terminal portions of the secondary DW-ACP.

Y ” _q is the non-target of the primary amplification product, excluding the binding site for the nucleotide sequence of sense opposite to the secondary DW-ACP of Formula II, as well as the nucleotide sequence complementary to the secondary DW-ACP Also provided is a regulator site that inhibits annealing of the X ″ _p site in the sequence. In addition, surprisingly, a modulator site located between two annealing sites, X ” _p and C _c , can provide high annealing specificity for both sites.

The 3′-terminal site, C _c , has a nucleotide sequence that hybridizes with the nucleotide sequence of sense opposite to the regulator site sequence of the secondary DW-ACP. Thus, when the regulator site of the secondary DW-ACP contains at least two deoxyinosine or inosine residues, as described above, the deoxycytidine nucleotide is most preferably inserted at the opposite site of the regulator site. Therefore, the 3'-terminal portion preferably comprises at least two deoxyguanosine nucleotides.

The description of the second DW-ACP may be applied to the third DW-ACP of the general formula III.

According to a preferred embodiment of the present invention, the method of the present invention removes the primary degenerate DW-ACP, the secondary DW-ACP and the primary target-specific primer before performing step (c). Further comprising (b) purifying the reaction product of step (a). For example, purification of the amplification products can be carried out by gel electrophoresis, column chromatography, affinity chromatography or hybridization. Most preferably, purification is carried out using a spin column with a silica-gel membrane. According to this method, the amplification products are adsorbed onto the silica-gel membrane in the presence of high salts, and impurities such as primers pass through the column. Thus, the amplification product can be obtained by rapid purification from the amplification reaction.

According to a more preferred embodiment of the invention, the method comprises a first amplification reaction step (a) and a second amplification reaction step (c) comprising a two-step amplification process. Most preferably, the method comprises a first amplification reaction step (a), a purification step (b) and a second amplification reaction step (c) comprising a two-step amplification process.

If the desired result of overcoming the non-specific amplification reaction is not achieved or suspected, or if the amplification product of step (c) is not visible in the agarose gel, amplify the inner region of the second amplification product. Another target-specific primer designed can be used to perform additional nested amplification reactions for at least one cycle.

The method of the present invention can be combined with other procedures known for specific purposes. For example, separation (or purification) of the amplification products may be followed by a first amplification or a second amplification reaction. 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 in a carrier for cloning. In addition, the amplification products of the present invention can be expressed in a suitable host having an expression vector. To express an amplification product, an expression vector is prepared that carries an amplification product under the control of the promoter or operably linked to the promoter. For standard expression of proteins or peptides in various host-expression systems, many standard techniques are known for constructing expression vectors containing amplification products and transcriptional / translational / regulatory sequences. Promoters for prokaryotic hosts include, but are not limited to, the pL λ promoter, trp promoter, lac promoter and T7 promoter. Promoters for eukaryotic hosts include, but are not limited to, metallothionein promoters, adenovirus late promoters, vaccinia virus 7.5K promoters, and promoters derived from polyoma, adenovirus 2, simian virus 40 or cytomegalovirus It doesn't happen. Examples of prokaryotic hosts include E. coli , Bacillus subtilis, and enterobacteria such as Salonella typhimurium, Serratia marsense and various Pseudomonas species. As well as microorganisms, cell cultures derived from multicellular organisms can be used as hosts. Cell cultures derived from vertebrates or invertebrates may be used. Mammalian cells, as well as insect cell systems infected with recombinant viral expression vectors (eg, baculovirus); And plants infected with a recombinant viral expression vector (eg, coliflower mosaic virus, tobacco mosaic virus) or transformed with a recombinant plasmid expression vector (eg, Ti plasmid) comprising one or more coding sequences can also be used. . Polypeptides expressed from amplification products can be purified according to methods known in the art.

According to another aspect of the present invention, the present invention provides a DNA working annealing regulatory primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, wherein the primer is represented by the following general formula (I). Provide a primer:

5'-X _p -Y _q -Z _r -Q _s -3 '(I)

Wherein X _p represents a 5′-terminal site having a pre-selected nucleotide sequence, and Y _q represents a regulatory site comprising at least two universal bases or non-differential base analogs, wherein Z _r Represents a degenerate random sequence site having a degenerate random nucleotide sequence, Q _s represents a 3′-terminal site having a hybridizing nucleotide sequence substantially complementary to one position of the unknown nucleotide sequence, p, q, r and s is the number of nucleotides; X, Y, Z and Q are deoxyribonucleotides or ribonucleotides.

According to another aspect of the present invention, the present invention provides a DNA working annealing control primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, wherein the primer is represented by the following general formula (II). Provide regulatory primers:

5'-X ' _p -S _u -Y' _v -Z ' _w -3' (II)

In the general formula, X ' _p represents a 5'-terminal portion having a nucleotide sequence corresponding to the 5'-terminal portion of the primary degenerate DW-ACP, S _u of step (a-2-1) target-specific primers represents the extension product of the first degeneracy DW-ACP regulator portion of the secondary annealing region comprising a nucleotide sequence that hybridizes to the other end portion for, Y _'v is the first degeneracy DW- ratio than the complementary nucleotide sequence to the ACP - the target sequence X 'and _p S _u A modulator site comprising at least two universal bases or non-distinguishing base analogs that interfere with the site being annealed, Z ' _w corresponds to the 3'-terminal site of the primary degenerate DW-ACP A 3'-terminal site having a nucleotide sequence, wherein p, u, v and w are the number of nucleotides; X ', S, Y', and Z 'are deoxyribonucleotides or ribonucleotides.

According to another aspect of the present invention, the present invention provides a DNA working annealing regulatory primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, wherein the primer is represented by the following general formula III. Provide a primer:

5'-X ” _p -Y” _q -C _c -3 '(III)

Wherein X ″ _p represents a 5′-terminal portion having a nucleotide sequence corresponding to all or a portion of the 5′-terminal portion of the secondary DW-ACP, and Y ″ _q represents a second of Formula II At least two of the secondary annealing site, a portion of the 5'-terminal site, a portion of the secondary annealing site and a portion of the 5'-terminal site, or a portion of the secondary annealing site and the regulator site of the primary DW-ACP; Annealing the X ″ _p site to a non-target sequence of a primary amplification product, excluding a nucleotide sequence complementary to a second DW-ACP, indicating a regulator site comprising a universal base or a non-specific base analog act to interfere with, and, C _c is the second DW-ACP of the 3'-end region and the regulator portion opposite with respect to all or a portion of the sequence - 3 ' end of the sense nucleotide sequence has a nucleotide sequence which hybridizes with the It denotes a portion, p, q, and c is the number of nucleotides, X ", Y", and C is a deoxyribonucleotide or a ribonucleotide.

Since the DW-ACP of the present invention is used in the amplification process of the present invention described above, the common content between them is omitted in order to avoid the complexity of the present specification that results in excessive repetitive description.

According to still another aspect of the present invention, the present invention provides a matrix comprising a primary degenerate DNA working annealing regulatory primer, a secondary DNA working annealing regulatory primer, a tertiary DNA working annealing regulatory primer, or a combination thereof. A kit is provided for amplifying an unknown nucleotide sequence adjacent to a nucleotide sequence.

According to a preferred embodiment of the invention, the kit of the invention may further comprise a target-specific primer. Optionally, the kits of the present invention may comprise reagents required for PCR reactions such as buffers, DNA polymerases, DNA polymerase cofactors and deoxyribonucleotide-5′-triphosphates. Optionally, the kits of the present invention may include antibodies that inhibit the activity of various polynucleotide molecules, reverse transcriptases, various buffers and reagents, DNA polymerases. The optimal amount of reagent used in a particular reaction can be readily determined by one of ordinary skill in the art having knowledge of the subject matter herein. Typically, the kit of the present invention comprises the aforementioned components in a separate package or compartment.

The present invention may provide an improved method of selectively amplifying an unknown nucleotide sequence from a nucleic acid or a mixture of nucleic acids (DNA or mRNA) by performing a nucleic acid amplification reaction, preferably PCR.

According to another aspect of the present invention, the present invention provides the use of the method of the invention described above for a process comprising nucleic acid amplification of an unknown nucleotide sequence adjacent to a known nucleotide sequence.

The present invention can be applied to various nucleic acid amplification-based techniques. Representative examples include:

(i) Genome walking to obtain unknown contiguous DNA sites on either side of the chromosomal site of a known nucleotide sequence. Examples include genome sequencing projects, cloning of promoter regions, identification of gene structures such as exon / intron junctions, gap filling and location or orientation of transgenes;

(Ii) rapid amplification (RACE) at the 5′- and 3′-ends of cDNA for cloning or sequencing of full-length cDNA, or splicing analysis;

(Iii) mapping of sites including deletions, insertions, and loci; And

(Iii) Rapid amplification of BAC ends without shotgun cloning for full genome sequencing.

The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the invention as determined by the appended claims.

1A is a diagram showing one specific embodiment of the primary amplification process using a primary degenerate DNA working annealing regulatory primer (DW-ACP), a secondary DW-ACP, and a primary target-specific primer. .

FIG. 1B shows a specific embodiment of a second amplification process using a third DW-ACP and a second target-specific primer.

2 shows amplification products generated by DNA working amplification. Conventional DNA working primers alone produced amplification products (lanes 1 and 2). In contrast, DW-ACP alone did not form any visible product (lane 4), but when tail primers were used, nonspecific products were formed due to nonspecific binding to the template of the tail primer (lane 3).

3 shows the amplification products generated by DW-ACP-amplification for amplification of the promoter sequence of the TNF-α gene. The main products of different sizes are different primary DW-ACPs, DW-ACP1-A (lane 1), DW-ACP1-T (lane 2), DW-ACP1-G (lane 3) and DW-ACP1- It is generated from C (lane 4). These products were identified as promoter sequences of the TNF-α gene by sequence analysis.

4 shows the amplification products generated by DW-ACP-amplification for amplification of the 5′-terminal region sequence of PBP cDNA. The main products of different sizes are different primary DW-ACPs, DW-ACP1-A (lane 1), DW-ACP1-T (lane 2), DW-ACP1-G (lane 3) and DW-ACP1- It is generated from C (lane 4).

Example

In the following examples, the following abbreviations are used: M (molar), mM millimolar), μM (micromolar), g (gram), μg (micrograms), ng (nanograms), L (liters), ml (milliliters) ), Μl (microliters), degree Centigrade; Roche (Roche Diagnostics, Mannheim, Germany); Promega (Promega Co., Madison, USA); QIAGEN (QIAGEN GmbH, Hilden, Germany); And Applied Biosystems (Foster City, CA, USA).

Oligonucleotide sequences used in the examples are listed in the Sequence Listing.

Example  One: DNA walking ACP - PCR  In the way ACP  Evaluation of the system

In order to evaluate the effect of the ACP system on DNA working ACP-PCR, the ACP system was compared with the conventional long primer system. Plant genome DNA was used as a template.

A. Primer Design

To amplify unknown sequences adjacent to sites of known sequences from DNA or nucleic acid mixtures of organisms, DNA working annealing regulatory primers (DW-ACPs) were designed. The primary DW-ACP was designed to have a trisegated structure with a polydioxyinosine [poly (dI)] linker between the 3'-terminal target binding sequence and the 5'-terminal non-target tail, wherein the 3'- The terminal target binding sequence comprises a predetermined abitaric sequence at the 3'-end and a degenerate random sequence at the 5'-end, with an A, between the 3'- and 5'-ends of the 3'-terminal target binding sequence; Any one of C, T and G was included. Thus, at least four DW-ACP primers are generated, depending on the aviary sequence at the 3'-end of the 3'-terminal target binding sequence of the DW-ACP.

Four primary degenerate DNA working annealing regulatory primers (primary DW-ACPs) were designed as follows, annealing regulatory primers (ACPs) were developed by the inventors and are disclosed in WO 03/050305:

DW-ACP1-A: 5'-TCACAGAAGTATGCCAAGCGAIIIINNNAGGTC-3 '(SEQ ID NO: 1);

DW-ACP1-C: 5'-TCACAGAAGTATGCCAAGCGAIIIINNNCGGTC-3 '(SEQ ID NO: 2);

DW-ACP1-T: 5'-TCACAGAAGTATGCCAAGCGAIIIINNNTGGTC-3 '(SEQ ID NO: 3); And

DW-ACP1-G: 5'-TCACAGAAGTATGCCAAGCGAIIIINNNGGGTC-3 '(SEQ ID NO: 4).

Secondary DNA working annealing regulatory primers (secondary DW-ACPs) were designed as follows:

DW-ACP2-N: 5'-TCACAGAAGTATGCCAAGCGAGGGGIIIIGGTC-3 '(SEQ ID NO: 5);

DW-ACP2-NA: 5'-TCACAGAAGTATGCCAAGCGAGGGGIIIAGGTC-3 '(SEQ ID NO: 6);

DW-ACP2-NC: 5'-TCACAGAAGTATGCCAAGCGAGGGGIIICGGTC-3 '(SEQ ID NO: 7);

DW-ACP2-NT: 5'-TCACAGAAGTATGCCAAGCGAGGGGIIITGGTC-3 '(SEQ ID NO: 8); And

DW-ACP2-NG: 5'-TCACAGAAGTATGCCAAGCGAGGGGIIIGGGTC-3 '(SEQ ID NO: 9).

Third DNA Working Annealing Control Primers (Third DW-ACPs) were designed as follows:

DW-ACP3-N1: 5'-TCACAGAAGTATGCCAAGCGAIIIIGGGGGGTC-3 '(SEQ ID NO: 10);

DW-ACP3-N2: 5'-TCACAGAAGTATGCCAAGCGAGIIIIGGGGGTC-3 '(SEQ ID NO: 11); And

DW-ACP3-N3 5'-TCACAGAAGTATGCCAAGCGAGGIIIIGGGGTC-3 '(SEQ ID NO: 12).

Another third DW-ACP was designed for nested primers to be used in nested DW-ACPs and the sequence is as follows:

Nested DW-P3-N: 5'-CCAAGCGAGGGGGGGGGGTC-3 '(SEQ ID NO: 13).

The polydioxyinosine [poly (dI)] linker of ACP inhibits annealing of the 5'-terminal tail sequence at the non-target position of the template, and causes primer hybridization at the 3'-end to the target sequence at a specific temperature. To increase the annealing specificity (Hwang et al., 2003).

As a control, conventional DNA working field primers (DW-Ps) with tails sequenced without a polydioxyinosine [poly (dI)] linker were used, whose sequence is as follows:

DW-P1-A: 5'-TCACAGAAGTATGCCAAGCGANNNAGGTC-3 '(SEQ ID NO: 14);

DW-P1-C: 5'-TCACAGAAGTATGCCAAGCGANNNCGGTC-3 '(SEQ ID NO: 15);

DW-P1-T: 5'-TCACAGAAGTATGCCAAGCGANNNTGGTC-3 '(SEQ ID NO: 16);

DW-P1-G: 5'-TCACAGAAGTATGCCAAGCGANNNGGGTC-3 '(SEQ ID NO: 17); And

DW-P1: 5'-TCACAGAAGTATGCCAAGCGANNNNGGTC-3 '(SEQ ID NO: 18).

The tail primers, JYC3 and JYC3-ACP, corresponding to the 5′-terminal region sequence of DW-ACPs and DW-Ps are as follows:

JYC3: 5'-TCACAGAAGTATGCCAAGCGA3 '(SEQ ID NO: 19).

Primers in this example were synthesized according to standard protocols using a DNA synthesizer (Expedite 8900 Nucleic Acid Synthesis System, Applied Biosystems (ABI)). Dioxyinosine in the primer was inserted using deoxyinosine CE phosphoramidite (ABI). Primers were purified using an OPC cartridge (ABI) and the concentration of the primers was determined at 260 nm with a UV spectrophotometer. In primers, “I” means deoxyinosine, and “N” means degenerate nucleotides occupied by any one of four deoxyribonucleotides, ie dATP, dCTP, dTTP and dGTP.

B. Genome DNA Isolation

Soybean chute was used for genome DNA isolation. The sample was rapidly frozen in liquid nitrogen and then ground. 200 μg of the ground sample was 15 ml containing 1.2 ml of digestion buffer (100 mM NaCl, 10 mM Tris-Cl pH 8.0, 25 mM EDTA pH 8.0, and 0.5% SDS) and 20 μl of protease K 6 μl. Was added to the tube. The tube was heated at 50 ° C. for 12 hours. Genome DNA was extracted twice with the same volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) and then once with chloroform / isoamyl alcohol (24: 1). This isolated genome DNA was used as a template to evaluate the effect of the ACP system in the DNA working ACP-PCR method.

C. First DNA Working PCR

Conventional genome working methods have a high background problem because DNA working (DW) primers non-specifically prime the template during PCR. Therefore, DNA walking PCR was performed using conventional DW-P alone or DW-ACP alone to determine whether DW primer alone produced a nonspecific product.

Conventional PCR for DW-P was performed without target-specific primers, 100 ng of soybean genome DNA, 5 μl of 10 × PCR buffer (Roche), 4 μl of dNTP (2.5 mM each of dATP, dCTP, dGTP, dTTP), 50 μl of reaction solution containing 5 μl of DW-P1 (10 μM) or 5 μl of DW-P1-C (10 μM), and 0.5 μl of Taq polymerase (5 units / μL, Roche); Placing the tube containing the reaction mixture in a preheated (94 ° C.) temperature circulator; The following PCR reaction was performed. 5 minutes denaturation at 94 ° C. followed by 30 cycles of 94 ° C. 40 seconds, 47 ° C. 40 seconds and 72 ° C. 60 seconds, followed by extension reaction at 72 ° C. for the final 5 minutes.

ACP-PCR for the first DW-ACP was performed by two-step PCR amplification to maximize the benefits of the ACP system.

Two-step PCR amplification was performed at two different annealing temperatures without target-specific primers, 100 ng soybean genome DNA, 5 μl of 10 × PCR buffer (Roche), dNTP (2.5 mM each for dATP, dCTP, dGTP, dTTP). 50 μl of reaction solution containing 4 μl, DW-ACP1-C (10 μM), and 0.5 μl Taq polymerase (5 units / μl, Roche) were used; Placing the tube containing the reaction mixture in a preheated (94 ° C.) temperature circulator; A first step PCR consisting of 94 ° C. 5 minutes, 45 ° C. 3 minutes and 72 ° C. 1 cycle was performed, followed by 30 cycles of 94 ° C. 40 seconds, 60 ° C. 40 seconds and 72 ° C. 60 seconds, followed by 72 ° C. The second step PCR was performed by extending the reaction for the last 5 minutes.

D. Purification of the Primary DNA Working Product

In order to remove primers such as DW-Ps or primary DW-ACPs used in the primary DNA working PCR, the primary amplification reaction product was purified using a spin column (PCR purification Kit, QIAGEN).

E. First DNA Working Secondary Amplification of PCR Products

To amplify the degenerate DNA working product, DW-P, primary DW-ACP or first, which selectively anneals only to sequences that are perfectly complementary to the entire pool of degenerate primary DW-ACPs. PCR was performed using tail primers (JYC3) corresponding to the 5'-terminal region sequence of the secondary DW-ACP.

The PCR reaction was carried out without target-specific primers, 2 μl of PCR product generated by DNA working PCR, 5 μl of 10 × PCR buffer (Roche), 4 μl of dNTP (2.5 mM each of dATP, dCTP, dGTP, dTTP), 50 μl of reaction solution containing 1 μl of tail primer JYC3 (10 μM) or 1 μl of DW-ACP2-N (10 μM) and 0.5 μl of Taq polymerase (5 units / μl, Roche); Placing the tube containing the reaction mixture in a preheated (94 ° C.) temperature circulator; The following PCR reaction was performed. 5 minutes denaturation at 94 ° C. followed by 30 cycles of 94 ° C. 40 seconds, 60 ° C. 40 seconds and 72 ° C. 60 seconds, followed by extension at 72 ° C. for the final 5 minutes.

F. Electrophoresis

Amplification products were detected by electrophoresis on 2% agarose gel and then stained with ethidium bromide.

2 shows amplification products of DNA working PCR performed using conventional DW primers or DW-ACP alone. Conventional DW primers, DW-P1 (lane 1) or a combination of DW-P1-C (lane 2) and JYC3, have generated many PCR products, even though no target-specific primers were used. The results show that conventional DW primers and tail primers produce non-specific products. The DW-ACP1-C and JYC3 combinations produced PCR products, even though no target-specific primers were used (lane 3), but the DW-ACP1-C and DW-ACP2-N combinations did not produce any visible products. Not formed (lane 4). These experimental results indicate that the primary DW-ACP can be used in the primary DNA working ACP-PCR, but if the tail primer JYC3 is used in the secondary PCR reaction, it binds to the nonspecific position of the template and eventually the nonspecific product. To amplify (lane 3). Conversely, when DW-ACP2-N is used in the secondary PCR reaction, this primer does not bind to the nonspecific position of the template due to the characteristics of the ACP system and eventually produces no product (lane 4).

Three important conclusions can be drawn from the above experimental results: 1) Conventional DW primers produce many nonspecific products in the first amplification because they are nonspecifically primed, and the resulting products are produced in the second amplification process. Amplified by tail primers; 2) In contrast, the first DW-ACP binds only to the specific position of the template in one cycle of first amplification (first stage PCR) under low stringency conditions, and this primer binds to the annealing site (ie, degenerate sequence and During the next cycle of primary amplification (second stage PCR) under high salt conditions where the 3'-terminal) cannot bind to the template or primary DW-ACP primer extension, it no longer acts as a primer, Thus, the second DW-ACP alone does not form any product in the second PCR reaction; And 3) when tail primers are used in the secondary PCR, the tail primers bind to the nonspecific position of the template to produce a nonspecific product.

The results in FIG. 2 show that the DNA working ACP-PCR system fundamentally solves the background problem, which is a significant problem of the prior art using tailing sequences, adapter sequences or cassette sequences as primers.

Example  2: DNA walking ACP PCR  From mouse genome using method TNF Amplification of the promoter sequence of the -α gene

In order to demonstrate the application of the DNA working ACP-PCR method of the invention, the promoter sequence of the mouse TNF-α gene was amplified from the mouse genome using TNF-α sequence information.

A. Primer Design

DW-ACPs designed in the same manner as in Example 1 were used for DNA working ACP-PCR amplification. Target-specific primers of the TNF-α gene were designed as follows:

mTNFα-C1: 5'-CACCTTGCCCTGCCCATTAG-3 '(SEQ ID NO: 20);

mTNFα-C2: 5'- CCCTCACACTGTCCTTCTTGCC-3 '(SEQ ID NO: 21);

mTNFα-C3: 5'-GAATAAGGGTTGCCCAGACACTC-3 '(SEQ ID NO: 22); And

mTNFα-C4: 5′-GGAGTGCCTCTTCTGCCAGTTC-3 ′ (SEQ ID NO: 23).

The primers were synthesized using a DNA synthesizer as in Example 1.

B. Genome DNA Isolation

As in Example 1, genome DNA was isolated from mouse placental tissue.

C. First DNA Working ACP PCR

The primary DNA working ACP PCR was performed in four separate tubes, each tube containing each primary DW-ACP, and the PCR was subjected to two-step PCR amplification to maximize the benefits of the ACP system. The ACP system was developed by the inventor and disclosed in WO 03/050305.

Two-step PCR amplification was performed at different 2 annealing temperatures, 100 ng of mouse genome DNA, 5 μl of 10 × PCR buffer (Roche), 4 μl of dNTP (2.5 mM each of dATP, dCTP, dGTP, dTTP), primary 1 μl of any of the DW-ACPs (10 μM), 1 μl of the second DW-ACP, 1 μl of DW-ACP2-N (10 μM), 1 μl of the primary target-specific primer mTNFα-C1 (10 μM), And 50 µl of the reaction solution containing 0.5 µl of Taq polymerase (5 units / µl, Roche); Placing the tube containing the reaction mixture in a preheated (94 ° C.) temperature circulator; Perform a first stage PCR consisting of 94 ° C 5 minutes, 40-45 ° C 1 minute and 72 ° C 1/2 cycle, followed by 10-30 cycles of 94 ° C 40 seconds, 55-60 ° C 40 seconds and 72 ° C 60 seconds Next, the second reaction was performed by extending the final reaction at 72 ° C. for 5 minutes.

D. First DNA Working ACP Purification of PCR Products

In order to remove primers such as DW-ACPs and primary target-specific primers used in the primary DNA working ACP PCR, the primary amplification reaction products were purified using spin column (PCR purification Kit, QIAGEN). It was.

E. Second DNA Working ACP PCR

To amplify only the unknown target-specific product from the primary DNA working ACP-PCR product, which may have a nonspecific product resulting from a nonspecific template for the template of the gene-specific primer mTNFα-C1, -ACP PCR was performed using a third DW-ACP and tested TNFα gene-specific primers, which were designed to amplify the internal site of the primary amplification product. The second PCR amplification was carried out 1-5 μl of the first PCR amplification product, 5 μl of 10 × PCR buffer (Roche), 4 μl of dNTP (2.5 mM each of dATP, dCTP, dGTP, dTTP), and the third DW-ACP DW 50 μl of reaction solution containing 1 μl of ACP3-N (10 μM), 1 μl of nested gene-specific primer mTNFα-C2 (10 μM), and 0.5 μl of Taq polymerase (5 units / μl, Roche) Used; Placing the tube containing the reaction mixture in a preheated (94 ° C.) temperature circulator; The following PCR reaction was performed. 5 minutes denaturation at 94 ° C. followed by 20-35 cycles of 94 ° C. 40 seconds, 55-60 ° C. 40 seconds and 72 ° C. 60 seconds, followed by extension reaction at 72 ° C. for the final 5 minutes.

If the secondary amplification product was not visible in the agarose gel, the third amplification was further performed using another nested target-specific primer designed to amplify the internal site of the secondary amplification product. The third amplification was performed by 1-2 μl of the second PCR amplification product, 5 μl of 10 × PCR buffer (Roche), 5 μl of 25 mM MgCl _2, 4 μl of dNTP (dATP, dCTP, dGTP, dTTP), 1 μl of 3rd DW-ACP DW-ACP3-N1 (10 μM), 1 μl of nested gene-specific primer mTNFα-C3 (10 μM), and 0.5 μl of Taq polymerase (5 units / μL, Roche) 50 µl of the reaction solution was used; Placing the tube containing the reaction mixture in a preheated (94 ° C.) temperature circulator; The following PCR reaction was performed. 5 minutes denaturation at 94 ° C. followed by 30-35 cycles of 94 ° C. 40 seconds, 60-65 ° C. 40 seconds and 72 ° C. 60 seconds, followed by extension reaction at 72 ° C. for the final 5 minutes.

F. Gel Extraction

Amplification products were detected by electrophoresis on 2% agarose gel and then stained with ethidium bromide. PCR products can also be detected by autoradiography or non-radioactive detection methods on modified polyacrylamide gels. After electrophoresis on agarose gels stained with EtBr, each PCR product was extracted using GENECLEAN II kit (Q-BIOgene, USA).

G. Cloning or Sequencing

The extracted fragments were cloned into pGEM-T Easy vector (Promega, USA) according to the manufacturer's protocol. Plasmids were transformed into XL1-Blue Compound cells. Transformed cells were plated on LB / ampicillin agar plates. Plasmids were separated from the white colonies alone. Insertion sequences were confirmed by EcoRI restriction enzyme treatment. Plasmids with insertion sequences were sequenced using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, USA). Alternatively, the extracted fragment was used as a template for direct sequencing and sequenced with an ABI PRISM 310 Genetic Analyzer (Perkin Elmer) using the BigDye Terminator cycle sequencing kit (Perkin Elmer). Computer-assisted sequence analysis was performed using the GeneRunner program (Hastings, Inc.).

Figure 3 shows the amplification products generated by DNA working ACP PCR for amplification of the promoter sequence of the TNF-α gene. The main products of different sizes are different primary DW-ACPs, DW-ACP1-A (lane 1), DW-ACP1-T (lane 2), DW-ACP1-G (lane 3) and DW-ACP1- It is generated from C (lane 4). These products were identified as promoter sequences of the TNF-α gene by sequence analysis. These experimental results indicate that the DNA working ACP PCR method of the present invention can be applied to amplify an unknown genome sequence adjacent to a known sequence of genomes.

Example  3: DNA walking PBP  Amplification of the 5′-terminal region sequence of cDNA

The full-length cDNA sequence of mouse preserinin-binding protein (PBP) is already known, but the DNA working technique of the present invention is used to amplify the 5'-terminal region sequence of PBP cDNA using only its 3'-terminal region sequence information. Applied.

A. Primer Design

DW-ACPs designed in the same manner as in Example 1 were used for DNA working ACP-PCR amplification. Target-specific primers of the mouse PBP gene were designed as follows based on their 3'-terminal region sequence information:

mPBP-C1: 5'-TCCACACCTTGAAGTCAAAGTC-3 '(SEQ ID NO: 24);

mPBP-C2: 5'-CAGAGGACAGGTACTGGACAGTAG-3 '(SEQ ID NO: 25); And

mPBP-C3: 5'-CAGTCTCATCACCATCCAGTCTC-3 '(SEQ ID NO: 26).

B. Primary cDNA Chain Synthesis

Total RNAs were isolated from the brain tissue of mouse strain ICR, and primary cDNA chains were synthesized using reverse transcriptase according to conventional methods (Hwang et al., 2000). Reverse transcription reactions were performed in 20 μl reaction solution containing the following components at 42 ° C. for 1.5 hours using total RNAs: 3 μg total RNA, 4 μl 5 × reaction buffer (Promega, USA), dNTPs (2 mM each) 5 μl, 2 μl 10 μM cDNA synthesis primer (dT ₂₀ or random hexamer), 0.5 μl RNase inhibitor (40 units / μl, Promega), and 1 μl reverse transcriptase (200 units / μl; Promega). The primary cDNAs were diluted by the addition of 80 μl of the first purified H ₂ O.

The sequence of the cDNA synthesis primer is as follows:

dT ₂₀ : 5'-TTTTTTTTTTTTTTTTTTTT-3 '(SEQ ID NO: 27); And

N ₆ : 5'-NNNNNN-3 '(SEQ ID NO: 28);

C. First DNA Working ACP PCR

The primary DNA working ACP PCR was carried out as in Example 2 above in four respective tubes containing each primary DW-ACP, but using the primary target-specific primer mPBP-C1.

D. First DNA Working ACP Purification of PCR Products

In order to remove primers such as primary DW-ACPs and primary target-specific primers used in the DNA working ACP PCR, the primary PCR product was purified using a spin column (PCR purification Kit, QIAGEN). It was. The purified primary amplification product may be diluted 1-10 fold depending on the result of the primary amplification, to be used as a template in subsequent secondary amplifications.

E. Second DNA Working ACP PCR

To amplify only the unknown target-specific product from the primary DNA working ACP-PCR product, which may have a nonspecific product resulting from a nonspecific template for the template of the gene-specific primer mPBP-C1, the second DW- ACP PCR was performed using a third DW-ACP and tested PBP gene-specific primers, which were designed to amplify the internal site of the primary amplification product. Secondary PCR reaction conditions were the same as in Process E of Example 2, but mPBP-C2 was used as the nested target-specific primer.

If the secondary amplification product was not visible in the agarose gel, the third amplification was further performed using another nested target-specific primer designed to amplify the internal portion of the secondary amplification product. The secondary amplification product can be diluted 1-1,000 fold depending on the result of the primary amplification, to be used as a template in subsequent tertiary amplifications. The third PCR reaction conditions were the same as the second ACP PCR, but mPBP-C3 was used as the nested target-specific primer.

F. Gel Extraction

Amplification products were analyzed in the same manner as described for gel extraction (process F) of Example 2.

G. Cloning or Sequencing

The amplification products were cloned or directly sequenced as described in Cloning or Sequencing Example 2 (Process G).

4 shows the amplification products generated by DNA working ACP PCR for amplification of the 5′-terminal region sequence of PBP cDNA. The main products of different sizes are different primary DW-ACPs, DW-ACP1-A (lane 1), DW-ACP1-T (lane 2), DW-ACP1-G (lane 3) and DW-ACP1- It is generated from C (lane 4). These products were identified by 5'-terminal region sequence of PBP cDNA through sequencing. These experimental results indicate that the DNA working ACP PCR method of the present invention can be applied to amplify unknown sequences downstream or upstream for some known cDNA sequences.

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

Reference

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Attach sequence list electronic file

Claims (30)

Primary amplification of an unknown nucleotide sequence using a DNA working annealing regulatory primer (DW-ACP) and a primary target-specific primer, said step (a) A method of amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence comprising the following process: (a-1) A primary degenerate DW-ACP comprising a degenerate random nucleotide sequence that hybridizes to said unknown nucleotide sequence and a hybridizing nucleotide sequence complementary to one location of said unknown nucleotide sequence A first stage amplification of the unknown nucleotide sequence at a first annealing temperature, comprising at least one cycle of primer annealing, primer extension and denaturation, wherein a first degenerate DW-ACP extension is produced. performing a stage amplification, wherein the first annealing temperature causes the first degenerate DW-ACP to act as a primer, (a-2) performing a second-stage amplification at a second annealing temperature that prevents the primary degenerate DW-ACP from acting as a primer, comprising: : (a-2-1) the primary degenerate DW-ACP extension using the primary target-specific primer hybridizing to a target-specific nucleotide sequence complementary to a position on the unknown nucleotide sequence A step of amplifying a target-specific primer extension product, (a-2-2) the target-specific primer extension using a second DW-ACP that hybridizes to a nucleotide sequence complementary to the primary degenerate DW-ACP sequence of the target-specific primer extension product Amplifying the product, whereby a second DW-ACP extension is produced, and (a-2-3) amplifying the second DW-ACP extension using the second DW-ACP and the first target-specific primer, by which step a degenerate random nucleotide A primary amplification product without sequence is produced. The method of claim 1 wherein the first stage amplification is performed one cycle. 2. The method of claim 1, wherein said second stage amplification is performed at least five cycles. The method of claim 1, wherein the primary annealing temperature is 35 ℃ to 50 ℃. The method of claim 1, wherein the second annealing temperature is 50 ℃ to 72 ℃. The method of claim 1 wherein the first degenerate DW-ACP is represented by the following general formula: 5'-X _p -Y _q -Z _r -Q _s -3 '(I) Wherein X _p represents a 5′-terminal site having a pre-selected nucleotide sequence, and Y _q represents a regulator site comprising 2-10 universal bases or non-differential base analogs, Z _r represents a degenerate random sequence site having a degenerate random nucleotide sequence, Q _s represents a 3′-terminal site having a hybridized nucleotide sequence complementary to one position of the unknown nucleotide sequence, p, q, r and s Is the number of nucleotides; X, Y, Z and Q are deoxyribonucleotides or ribonucleotides. 7. The method of claim 6, wherein said modulator site of said primary degenerate DW-ACP acts to define an annealing site to the 3'-terminal site of said primer at a first annealing temperature. The method of claim 6, wherein the second degenerate DW-ACP is represented by the following general formula II: 5'-X ' _p -S _u -Y' _v -Z ' _w -3' (II) In the general formula, X ' _p represents a 5'-terminal portion having a nucleotide sequence corresponding to the 5'-terminal portion of the primary degenerate DW-ACP, S _u of step (a-2-1) target-specific primers represents the extension product of the first degeneracy DW-ACP regulator portion of the secondary annealing region comprising a nucleotide sequence that hybridizes to the other end portion for, Y _'v is the first degeneracy DW- ratio than the complementary nucleotide sequence to the ACP - the target sequence X _'p and to prevent the annealing region S _u, 2-5 universal bases or non-represents a regulator portion comprising a distinctness nucleotide analogs, Z ' _w represents a 3'-terminal portion having a nucleotide sequence corresponding to the 3'-terminal portion of the primary degenerate DW-ACP, p, u, v and w are the number of nucleotides; X ', S, Y', and Z 'are deoxyribonucleotides or ribonucleotides. The method of claim 1, wherein the method comprises a nucleotide sequence that hybridizes with the opposite-sense nucleotide sequence for the second DW-ACP sequence present at the 3′-end of the primary amplification product to the 3′-end site. Primer annealing, primer extension with a third DW-ACP comprising and a nested target-specific primer for amplifying the primary target-specific primer of step (a) or an internal site of the primary amplification product And (c) conducting a secondary amplification at a third annealing temperature, comprising at least one cycle of denaturation. The method of claim 9, wherein the third annealing temperature is 50 ℃ to 72 ℃. 10. The method of claim 9, wherein the method comprises the steps of removing the primary degenerate DW-ACP, the secondary DW-ACP and the primary target-specific primer before performing step (c): and (b) purifying the reaction product of a). 10. The method of claim 9, wherein the third DW-ACP is represented by the following general formula III: 5'-X ” _p -Y” _q -C _c -3 '(III) Wherein X ″ _p represents a 5′-terminal portion having a nucleotide sequence corresponding to all or a portion of the 5′-terminal portion of the secondary DW-ACP, and Y ″ _q represents a second of Formula II Corresponds to the secondary annealing site, part of the 5'-terminal site, part of the secondary annealing site and part of the 5'-terminal site of the primary DW-ACP, or part of the secondary annealing site and the modulator site, 2-5 A regulator site comprising a universal base or a non- discriminatory base analog of the dog, wherein the X ″ _p site anneals to the non-target sequence of the primary amplification product, excluding the nucleotide sequence complementary to the secondary DW-ACP C _c is a 3'-terminal having a nucleotide sequence that hybridizes with the opposite-sense nucleotide sequence to all or a portion of the 3'-terminal and regulator region sequences of the secondary DW-ACP. part It represents a, p, q, and c is the number of nucleotides, X ", Y", and C is a deoxyribonucleotide or a ribonucleotide. The method of claim 1 wherein the nucleotide sequence to be amplified is gDNA or cDNA. 13. A compound according to any one of claims 6, 8 and 12, wherein the universal base or non- discriminatory base analog is deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza -2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2'-OMe 3-nitropyrrole, 2'-F 3- Nitropyrrole, 1- (2'-deoxy-beta-D-ribofuranosyl) -3-nitropyrrole, deoxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebulin, 2'- F nebulin, 2'-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, Morpholino-5-nitroindole, morpholino-nebulin, morpholino-inosine, morpholino-4-nitrobenz Midazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebulin, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole , Phosphoramidate-3-nitropyrrole, 2'-0-methoxyethylinosine, 2'-0-methoxyethyl nebulin, 2'-0-methoxyethyl 5-nitroindole, 2 ' -0-methoxyethyl 4-nitro-benzimidazole, 2'-0-methoxyethyl 3-nitropyrrole and a combination of said bases. 15. The method of claim 14, wherein the universal base or non-specific base analog is deoxyinosine, inosine, 1- (2'-deoxy-beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitro. Characterized in that it is an indole. 16. The method of claim 15, wherein said universal base or non- discriminatory base analog is deoxyinosine. 13. The method of any one of claims 6, 8 and 12, wherein said modulator site comprises a contiguous sequence of nucleotides having universal bases or non-differential base analogs. 13. The method of any one of claims 6, 8 and 12, wherein p is an integer from 10 to 60. delete 13. A method according to any one of claims 6, 8 and 12, wherein q or u is an integer from 2 to 10. 9. The method of claim 6 or 8, wherein r or v is an integer from 2 to 5. 9. A method according to claim 6 or 8, wherein s or w is an integer from 3 to 10. 13. The method of claim 12 wherein c is an integer from 5 to 15. 9. The method of claim 8, wherein S comprises 2-10 consecutive deoxyguanosine residues. 13. The method of claim 12, wherein C comprises 2-5 consecutive deoxyguanosine residues at positions that hybridize with the counter-sense nucleotide sequence to the regulator site sequence of the secondary DW-ACP How to. DNA working annealing regulatory primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, wherein said primer is represented by the following general formula (I): 5'-X _p -Y _q -Z _r -Q _s -3 '(I) Wherein X _p represents a 5′-terminal site having a pre-selected nucleotide sequence, and Y _q represents a regulator site comprising 2-10 universal bases or non-differential base analogs, Z _r represents a degenerate random sequence site having a degenerate random nucleotide sequence, Q _s represents a 3′-terminal site having a hybridized nucleotide sequence complementary to one position of the unknown nucleotide sequence, p, q, r and s Is the number of nucleotides; X, Y, Z and Q are deoxyribonucleotides or ribonucleotides. DNA working annealing regulatory primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, wherein said primer is represented by the following general formula II: 5'-X ' _p -S _u -Y' _v -Z ' _w -3' (II) In the general formula, X ' _p represents a 5'-terminal portion having a nucleotide sequence corresponding to the 5'-terminal portion of the primary degenerate DW-ACP, S _u of step (a-2-1) target-specific primers represents the extension product of the first degeneracy DW-ACP regulator portion of the secondary annealing region comprising a nucleotide sequence that hybridizes to the other end portion for, Y _'v is the first degeneracy DW- ratio than the complementary nucleotide sequence to the ACP - the target sequence X _'p and to prevent the annealing region S _u, 2-5 universal bases or non-represents a regulator portion comprising a distinctness nucleotide analogs, Z ' _w represents a 3'-terminal portion having a nucleotide sequence corresponding to the 3'-terminal portion of the primary degenerate DW-ACP, p, u, v and w are the number of nucleotides; X ', S, Y', and Z 'are deoxyribonucleotides or ribonucleotides. A DNA working annealing regulatory primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, wherein said primer is represented by the following general formula III: 5'-X ” _p -Y” _q -C _c -3 '(III) Wherein X ″ _p represents a 5′-terminal portion having a nucleotide sequence corresponding to all or a portion of the 5′-terminal portion of the secondary DW-ACP, and Y ″ _q represents a second of Formula II Corresponds to the secondary annealing site, part of the 5'-terminal site, part of the secondary annealing site and part of the 5'-terminal site of the primary DW-ACP, or part of the secondary annealing site and the modulator site, 2-5 A regulator site comprising a universal base or a non- discriminatory base analog of the dog, wherein the X ″ _p site anneals to the non-target sequence of the primary amplification product, excluding the nucleotide sequence complementary to the secondary DW-ACP C _c is a 3'-terminal having a nucleotide sequence that hybridizes with the opposite-sense nucleotide sequence to all or a portion of the 3'-terminal and regulator region sequences of the secondary DW-ACP. part It represents a, p, q, and c is the number of nucleotides, X ", Y", and C is a deoxyribonucleotide or a ribonucleotide. A kit for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence comprising the DNA working annealing regulatory primer of claim 26, the DNA working annealing regulatory primer of claim 27, the DNA working annealing regulatory primer of claim 28, or a combination thereof. . delete

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