Patent Application
20100221787
The present invention relates to an isothermal amplification method and a DNA polymerase used in the same.
DNA polymerases are some of the most widely used enzymes in the field of life science, and they are essential in various techniques including a polymerase chain reaction (PCR) method, for example. Many kinds of such DNA polymerases are commercially available, and each polymerase is characterized by the reaction conditions therefor and an enzyme activity(s) thereof, for example. The most well known DNA polymerase I from Escherichia coli has a 5′→3′ polymerase activity, which allows, from a template DNA and a primer, a complementary sequence to the template to be synthesized, and also has a 5′→3′ exonuclease activity and a 3′→5′ exonuclease activity. It is known that these three enzyme activities pertain to different structural domains. That is, they pertain to a 5′→3′ exonuclease domain on an N-terminal side, a 3′→5′ exonuclease domain in a central portion, and a polymerase domain on a C-terminal side, respectively (see Non-Patent Document 1, for example). A large fragment of 75 kD obtained by treating the DNA polymerase I with subtilisin is also called “Klenow fragment”, and it lacks the 5′→3′ exonuclease activity among the above-described three activities. Thus, the Klenow fragment is useful in a sequence reaction according to a dideoxy method, a reaction for blunting a 5′-protruding end, and the like. Currently, Klenow fragments expressed and purified as recombinant proteins of the DNA polymerase I with no small fragments on the N-terminal side are commercially available.
As a DNA amplification technique, a PCR method generally is used. However, the PCR method has problems in that complicated temperature control is required, a thermal cycler for conducting such complicated temperature control is required, it takes several hours to complete the reaction, etc. Thus, as a DNA amplification technique as an alternative to the PCR method, a LAMP (Loop-mediated Isothermal Amplification) method (see Non-Patent Document 2, for example), a SDA (Strand Displacement Amplification) method (see Patent Document 1, for example), a method proposed by Mitani et al. (see Patent Documents 4, 5, and 6, and Non-Patent Document 3, for example), and the like have been developed. These methods are called isothermal amplification methods because amplification reactions in these methods can be carried out isothermally. In these methods, complicated temperature control and a thermal cycler for conducting it as required in PCR are not necessary. On the other hand, a DNA polymerase having a complementary strand displacement replication activity (see Patent Documents 2 and 3, for example) is essential for the isothermal amplification reactions. Currently, only a few kinds of DNA polymerase having a complementary strand displacement replication activity are available on the market, and it has been pointed out that reaction conditions therefor such as an optimum temperature are limited, a reaction time is long, or the like. Such problems place restrictions on the development of test agents, diagnostic agents, etc. using these DNA amplification methods.
The DNA polymerases currently used in the isothermal amplification methods act at relatively low temperatures. Thus, in the amplification reactions, specificity in annealing between a template DNA and a primer is low. Accordingly, it has been pointed out that there are problems such that by-products are produced owing to the decreased specificity in the amplification reactions and that it is difficult to amplify a relatively long target sequence. Furthermore, even if the same DNA polymerase is used, for example, the amplification efficiency varies depending on the isothermal amplification method to which it is applied. Thus, DNA polymerases suitable for the respective isothermal amplification methods are demanded.
With the foregoing in mind, it is an object of the present invention to provide a DNA polymerase suitable for specific isothermal amplification methods and an isothermal amplification method using the same.
An isothermal amplification method of the present invention is an isothermal amplification method for carrying out isothermal amplification of a target nucleic acid sequence in a nucleic acid sample. The method includes carrying out an amplification reaction of the target nucleic acid sequence isothermally in the presence of a DNA polymerase composed of a protein described in any of the following items (a) to (d) using a first primer shown in the following (X):
(a) a protein having an amino acid sequence represented by SEQ ID NO. 23;
(b) a protein having an amino acid sequence represented by SEQ ID NO. 25;
(c) a protein having an amino acid sequence obtained by deletion of any number from 1 to 334 of consecutive amino acid residues starting from an N-terminal in the amino acid sequence represented by SEQ ID NO. 23;
(d) a protein having an amino acid sequence obtained by deletion, substitution, insertion, or addition of one or more amino acids in the amino acid sequence of the protein described in any of the items (a) to (c) and having a DNA polymerase activity; and
(X) a primer that contains, in a 3′ end portion, a sequence (Ac′) that hybridizes to a sequence (A) of a 3′ end portion of the target nucleic acid sequence and also contains, on a 5′ side of the sequence (Ac′), a sequence (B′) that hybridizes to a complementary sequence (Bc) to a sequence (B) present on a 5′ side with respect to the sequence (A) in the target nucleic acid sequence.
An isothermal amplification DNA polymerase according to the present invention is a DNA polymerase to be used in the isothermal amplification method of the present invention. The DNA polymerase is composed of a protein described in any of the following items (a) to (d).
An isothermal amplification kit of the present invention is an isothermal amplification kit to be used in the isothermal amplification method of the present invention. The kit includes the isothermal amplification DNA polymerase according to the present invention.
The inventors of the present invention found that thermostable Alicyclobacillus acidocaldarius-derived DNA polymerase (hereinafter referred to as “Aac polymerase”) having a DNA replication activity and a complementary strand displacement replication activity is suitable for specific methods to be described later among various isothermal amplification methods, and thus achieved the present invention. The isothermal amplification DNA polymerase of the present invention can improve the amplification efficiencies of the specific isothermal amplification methods to be described later as compared with conventional DNA polymerases used in the isothermal amplification methods. As a result, the isothermal amplification DNA polymerase of the present invention allows nucleic acid amplification to be carried out in a shorter time than ever before. If the time required for nucleic acid amplification can be shortened, for example, analysis of single nucleotide polymorphism (SNP) or the like utilizing the nucleic acid amplification can be conducted more rapidly and a large amount of specimen can be analyzed more efficiently. Therefore, the present invention is very useful in the fields of nucleic acid analyses, clinical tests, and the like utilizing nucleic acid amplification. The fact that the Aac DNA polymerase is suitable for the specific isothermal amplification methods is newly discovered by the inventors of the present invention.
The isothermal amplification DNA polymerase according to the present invention is, as described above, a DNA polymerase to be used in the isothermal amplification method of the present invention and composed of a protein described in any of the following items (a) to (d). The isothermal amplification method of the present invention to which the DNA polymerase of the present invention is applied will be described later.
(a) a protein having an amino acid sequence represented by SEQ ID NO. 23
(b) a protein having an amino acid sequence represented by SEQ ID NO. 25
(c) a protein having an amino acid sequence obtained by deletion of any number from 1 to 334 of consecutive amino acid residues starting from an N-terminal in the amino acid sequence represented by SEQ ID NO. 23,
(d) a protein having an amino acid sequence obtained by deletion, substitution, insertion, or addition of one or more amino acids in the amino acid sequence of the protein described in any of the items (a) to (c) and having a DNA polymerase activity
The isothermal amplification DNA polymerase according to the present invention can be isolated from, for example, microorganisms of the genus Alicyclobacillus, preferably from Alicyclobacillus acidocaldarius (A. acidocaldarius), and more preferably from A. acidocaldarius JCM 5260. This strain (JCM 5260) can be purchased from RIKEN (independent administrative institution) BioResource Center, Japan Collection of Microorganisms (http://www.jcm.riken.jp/JCM/Ordering_J.shtml). The isothermal amplification DNA polymerase according to the present invention hereinafter is also referred to as “DNA polymerase of the present invention”, “DNA polymerase I of the present invention”, “Aac polymerase of the present invention”, or “protein of the present invention”.
The DNA polymerase represented by SEQ ID NO. 23 in the item (a) can be isolated as a full-length DNA polymerase from A. acidocaldarius JCM 5260, for example. The DNA polymerase represented by SEQ ID NO. 25 in the item (b) is a protein having an amino acid sequence obtained by deletion of 1st to 334th amino acid residues starting from the N-terminal in the amino acid sequence represented by SEQ ID NO. 23 in the item (a). The protein having an amino acid sequence obtained by deletion of an N-terminal region formed of 1st to 334th amino acids in the amino acid sequence of SEQ ID NO. 23 as described above still has a DNA polymerase activity. Furthermore, the protein described in the item (c) is a protein having an amino acid sequence obtained by deletion of any number from 1 to 334 of consecutive amino acid residues starting from an N-terminal in the amino acid sequence represented by SEQ ID NO. 23. The protein having an amino acid sequence in which any number of amino acid residues are deleted in the above-described N-terminal region as described above still has a DNA polymerase activity. The number of the deleted amino acid residues is not particularly limited, and is, for example, in the range from 1 to 334. Furthermore, in the N-terminal region, consecutive amino acid residues may be deleted, or non-consecutive amino acid residues may be deleted.
As shown in the item (d), the DNA polymerase of the present invention may be a protein having an amino acid sequence obtained by deletion, substitution, insertion, or addition of one or more amino acids in the amino acid sequence described in any of the items (a) to (c), as long as it has a DNA polymerase activity. The expression “one or more amino acids” means, for example, about 5% to 10% of the number of the amino acid residues of the protein described in any of the items (a) to (c), and is, for example, about 1 to 50, preferably about 1 to 20, more preferably about 1 to 10, and most preferably about 1 to 5.
Furthermore, the DNA polymerase of the present invention may be a protein having a homology of, for example, at least 50% to the amino acid sequence of the protein described in any of the items (a) to (c) as shown in the following item (e), as long as it has a DNA polymerase activity. The homology preferably is at least 70%, at least 80%, at least 85%, at least 90%, at least 97%, and at least 98%.
(e) a protein having an amino acid sequence with a homology of, for example, at least 50% to the amino acid sequence of the protein described in any of the items (a) to (c) and having a DNA polymerase activity.
Usually, the degree of the homology of two proteins can be indicated as a percentage of the identity between amino acid sequences of the proteins when they are aligned appropriately, and it represents the occurrence ratio of perfect match between the amino acid sequences. The appropriate alignment between the sequences for comparison of the identity can be determined using one of various algorithms, for example, a BLAST algorithm (Altschul, S. F. et al., J. Mol. Biol., 1990, Vol. 215, No. 3, pp. 403-410).
The DNA polymerase of the present invention has, as polymerase activities, a template-dependent DNA replication activity and a complementary strand displacement replication activity, which are both ordinary activities, and it further has, for example, a reverse transcriptase activity. The DNA polymerase of the present invention further may have a 3′→5′ exonuclease activity. When the DNA polymerase of the present invention has a 3′→5′ exonuclease activity, the occurrence of error at the time of incorporating a substrate can be reduced further, for example. It is preferable that the DNA polymerase of the present invention exhibit an activity at, for example, any temperature from 25° C. to 75° C., more preferably from 37° C. to 72° C., still more preferably from 50° C. to 70° C., and particularly preferably from 55° C. to 65° C. The optimum temperature for the DNA polymerase of the present invention is, for example, higher than the optimum temperature (e.g., 20° C. to 37° C.) for known DNA polymerases having a complementary strand displacement replication activity. Specifically, for example, it is preferable that the DNA polymerase of the present invention exhibit the activity at any temperature from 25° C. to 75° C., more preferably from 37° C. to 72° C., still more preferably from 50° C. to 70° C., and particularly preferably from 55° C. to 65° C. Therefore, the DNA polymerase of the present invention can be used under reaction conditions with a temperature higher than that for the conventional DNA polymerases having a complementary strand displacement replication activity, for example. Thus, in specific isothermal amplification methods in the present invention including the SMAP method and LAMP method to be described later and the like, the DNA polymerase of the present invention can be used under more strict annealing conditions for a template DNA and a primer. Moreover, in the case where the DNA polymerase of the present invention has a reverse transcriptase activity, for example, it can be used for DNA synthesis using RNA as a template, and thus can be used in a method alternative to conventional RT-PCR.
Furthermore, the isothermal amplification DNA polymerase of the present invention includes, for example, an N-terminal deleted DNA polymerase in which an N-terminal side amino acid residue(s) is deleted as described above. The number of the N-terminal side amino acid residues to be deleted is not particularly limited, as long as, for example, the resultant N-terminal deleted DNA polymerase has a complementary strand displacement replication activity as described above. As a specific example, the N-terminal deleted DNA polymerase preferably is one that lacks a 5′→3′ exonuclease activity and has, for example, activities corresponding to those of Klenow fragment of DNA polymerase I from Escherichia coli. Such a DNA polymerase may be, for example, a protein having an amino acid sequence obtained by deletion of any number from 1 to 334 of consecutive amino acid residues starting from an N-terminal in the amino acid sequence represented by SEQ ID NO. 23, as shown in the item (b) or (c). Conceivably, when a relatively large number of consecutive amino acid residues starting from the N-terminal are deleted, the possibility that the DNA polymerase might lack the 5′→3′ exonuclease activity is relatively high. In DNA polymerases, a 5′→3′ exonuclease domain, a 3′→5′ exonuclease domain, and a polymerase domain are arranged in this order from the N-terminal side. Accordingly, when deleting the 5′→3′ exonuclease activity, this can be achieved by, for example, deleting any number of amino acid residues from the N-terminal side in the amino acid sequence of SEQ ID NO. 23 until the 5′→3′ exonuclease activity is no longer exhibited. In the amino acid sequence of SEQ ID NO. 23, a region formed of 62nd to 306th amino acids is considered to be a 5→3′ exonuclease domain and a region formed of 392nd to 536th amino acids is considered to be a 3→5′ exonuclease domain. However, the present invention is not limited thereto. As the isothermal amplification DNA polymerase according to the present invention, SEQ ID NO. 25 shows a specific example of the amino acid sequence of an N-terminal deleted DNA polymerase that has a DNA polymerase activity and a 3′→5′ exonuclease activity corresponding the activities of Escherichia coli Klenow fragment and lacks a 5′→3′ exonuclease activity. When the DNA polymerase of the present invention lacks the 5′→3′ exonuclease activity as described above, there is an advantage in that, during gene amplification, the amplification product obtained can be prevented from being degraded, for example.
Furthermore, the isothermal amplification DNA polymerase of the present invention may lack the 3′→5′ exonuclease activity. From the comparison of amino acid sequences of various kinds of DNA polymerase I, it has been known that, in the enzymes having the 3′→5′ exonuclease activity, three common sequence motifs are present in the central portion of the enzyme proteins. Enzymes from which these motifs are removed can be prepared. For example, commercially available Bst DNA polymerase derived from Bacillus stearothermophilus lacks the 3′→5′ exonuclease activity (Aliotta, J. M. et al., Genetic Analysis: Biomolecular Engineering, Vol. 12, pp. 185-195, 1996). The DNA polymerase lacking the 3′→5′ exonuclease activity does not degrade the 3′ end of a primer when it is used in an isothermal amplification reaction, for example. Thus, it brings about an advantage in that the reduction in primer concentration is prevented sufficiently, thus allowing a nucleic acid to be amplified more rapidly. Moreover, the DNA polymerase lacking the 3′→5′ exonuclease activity is suitable for use in, for example, the detection of point mutation. According to the DNA polymerase lacking the 3′→5′ exonuclease activity, it is possible to prevent sufficiently, for example, a primer from being degraded from its 3′ side. Thus, the primer can discriminate sufficiently the mutation site of the template DNA, so that, for example, erroneous proceeding of the extension reaction can be suppressed and the detection of point mutation based on the termination of the extension reaction can be performed more accurately. As described above, in DNA polymerases, the 5′→3′ exonuclease domain, the 3′→5′ exonuclease domain, and a polymerase domain are arranged in this order from the N-terminal side. Accordingly, when deleting the 3′→5′ exonuclease activity, this can be achieved by, for example, deleting any number of amino acid residues in the amino acid sequence of SEQ ID NO. 23 or SEQ ID NO. 25 until the 3′→5′ exonuclease activity no longer is exhibited with the DNA polymerase activity being maintained. Furthermore, in the amino acid sequence of SEQ ID NO. 23, a region formed of 392nd to 536th amino acids is considered to be a 3′→5′ exonuclease domain. Thus, for example, this region may be deleted.
On the other hand, it has been suggested that, for the expression of the 3′→5′ exonuclease activity, for example, two metal ions coordinated in a carboxyl group in a side chain of an amino acid as an active center are important. Thus, for example, even when a DNA polymerase has a low 3′→5′ exonuclease activity, the enzyme activity can be enhanced by introducing an amino acid having a carboxyl group in its side chain into the active center (Park, Y. et al., Mol. Cells. Vol. 7, No. 3, pp. 419-424, 1997).
A method for measuring the activities of the DNA polymerase is not limited, and they can be measured by various measurement methods well known to those skilled in the art. Among the DNA polymerase activities, the template-dependent DNA replication activity can be measured using a fluorometric measurement method described in the literature (Seville M. et al. Biotechniques Vol. 21, pp. 664-668 (1996)) or the like, for example. Furthermore, among the DNA polymerase activities, the complementary strand displacement replication activity can be measured by the measurement method described in, for example, the above-described
Non-Patent Document 1 (Kornberg, A. and Baker TA. DNA Replication, W.H. Freeman and Company, New York, 1992.) or the like. Still further, among the DNA polymerase activities, the reverse transcriptase activity can be measured by using, for example, a commercially available kit (e.g., EnzChek (trademark) Reverse Transcriptase Assay Kit (E-22064), Molecular Probes (Invitrogen)) in accordance with the protocol thereof.
The isothermal amplification method of the present invention is an isothermal amplification method for carrying out isothermal amplification of a target nucleic acid sequence in a nucleic acid sample. The method includes amplifying the target nucleic acid sequence isothermally in the presence of the DNA polymerase of the present invention composed of a protein described in any of the above-described items (a) to (d) using a first primer shown in the following (X).
P (X) a primer that contains, in a 3′ end portion, a sequence (Ac′) that hybridizes to a sequence (A) of a 3′ end portion of the target nucleic acid sequence and also contains, on a 5′ side of the sequence (Ac′), a sequence (B′) that hybridizes to a complementary sequence (Bc) to a sequence (B) present on a 5′ side with respect to the sequence (A) in the target nucleic acid sequence
The present invention provides the isothermal amplification method using the first primer shown in the above-described item (X). The method is characterized in that the isothermal amplification DNA polymerase of the present invention is used therein. Therefore, as long as the first primer shown in the item (X) and the isothermal amplification DNA polymerase according to the present invention are used in the method, other configurations, steps, conditions, etc. are not limited. Furthermore, according to the isothermal amplification reaction using the first primer shown in the item (X), a ladder-like amplification product is obtained at the time of electrophoretic analysis, for example. Thus, the isothermal amplification reaction to which the DNA polymerase of the present invention is applied also can be referred to as, for example, an isothermal amplification reaction for generating a ladder-like amplification product.
The isothermal amplification method generally is a method of carrying out a nucleic acid amplification reaction isothermally. The conditions for the amplification reaction are not particularly limited and can be determined as appropriate by those skilled in the art. Preferably, the reaction temperature is set at, for example, a temperature around the melting temperature (Tm) of the primer or lower. More preferably, the stringency level is set in view of the melting temperature (Tm) of the primer. Specific examples of the reaction temperature include about 20° C. to about 75° C., preferably about 37° C. to about 72° C., more preferably about 50° C. to 70° C., and still more preferably about 55° C. to 65° C.
When carrying out the amplification reaction, it is preferable to cause a mismatch binding protein to be present as well because it can improve specificity, for example. The mismatch binding protein (also referred to as a “mismatch recognition protein”) is not limited, as long as it is a protein capable of recognizing a mismatch in, for example, a double-stranded nucleic acid and binding to a mismatch site thereof. For example, proteins known to those skilled in the art can be used. Furthermore, the mismatch binding protein may be, for example, a protein (a mutant) having an amino acid sequence obtained by substitution, deletion, addition, and/or insertion of one or more amino acids in an amino acid sequence of a wild-type protein, as long as it can recognize the mismatch in, for example, a double-stranded nucleic acid. Many mismatch binding proteins are known including, for example, a MutS protein (e.g., JP 9 (1997)-504699 A), a MutM protein (e.g., JP 2000-300265 A), a MutS protein bonded to a green fluorescence protein (GFP) (WO 99/06591), Taq MutS, and analogs thereof (Radman, M. and Wagner, R., Annu. Rev. Genet. 20: 523-538 (1986); Radman, M. and Wagner, R., Sci. Amer., 1988, pp 40-46; Modrich, P., J. Biol. Chem. 264: 6597-6600 (1989); Lahue, R. S. et al., Science 245: 160-164 (1988); Jiricny, J. et al., Nucl. Acids Res. 16: 7843-7853 (1988); Su, S. S. et al., J. Biol. Chem. 263; 6829-6835 (1988); Lahue, R. S. et al., Mutat. Res. 198: 37-43 (1988); Dohet, C. et al., Mol. Gen. Gent. 206: 181-184 (1987); Jones, M. et al., Genetics 115:605-610 (1987); MutS of Salmonella typhimurium (Lu, A. L., Genetics 118: 593-600 (1988); Haber L. T. et al., J. Bacteriol. 170; 197-202 (1988); Pang, P. P. et al., J. Bacteriol. 163; 1007-1015 (1985)); Priebe S. D. et al., J. Bacterilo. 170: 190-196 (1988); and the like). In the present invention, preferable mismatch binding proteins include MutS, MSH2, MSH6, MutH, MutL, and one derived from yeast, for example.
Preferably, the aforementioned mismatch binding protein is being activated by an activator, for example, in order to prevent it from binding to a double-stranded nucleic acid containing no mismatch. The activator is not particularly limited. Examples thereof include ATP (adenosine 5′-triphosphate), ADP (adenosine 5′-diphosphate), ATP-γ-S (adenosine 5′-O-(3-thiotriphosphate)), and AMP-PNP (adenosine 5′-[β, γ-imide]triphosphate). Furthermore, the activator may be one of the nucleotides that can bind to a mismatch binding protein. A mismatch binding protein can be activated by incubating the mismatch binding protein and the activator at room temperature for several seconds to several minutes.
In the case where the mismatch binding protein is used, it is preferable to further use a single-stranded binding protein (SSB), for example, in order to prevent the mismatch binding protein from binding to a single-stranded nucleic acid. The SSB is not particularly limited and conventionally known proteins can be used. Specific examples of the SSB include single-stranded binding proteins derived from Escherichia coli, Drosophila, and Xenopus laevis, gene 32 proteins derived from T4 Bacteriophage, and in addition, those proteins derived from other species. In this case, examples of the mismatch binding protein include MutS, MutH, MutL, HexA, MSH1 to MSH6, Rep3, RNaseA, uracil-DNA glycosidase, T4 endonuclease VII, and resolvase. The mismatch binding protein is preferably MutS, MSH2, MSH6, or a mixture of two or more of them, and is more preferably MutS.
Examples of the isothermal amplification method using the first primer shown in the (X) include: LAMP (Loop-Mediated Isothermal Amplification) methods disclosed in WO 00/28082, Notomi, T et. al., Nucleic Acids Research (2000), Vol. 28, e63., and the like; and methods disclosed in Japanese Patent No. 3867926, Japanese Patent No. 3897805, Japanese Patent No. 3942627, and NATURE METHODS (Vol. 4, No. 3, March 2007, pp. 257-262), Mitani Y., Lezhava A., Kawai Y., Kikuchi T., Oguchi-Katayama A., Kogo Y., Itoh M., Miyagi T. et al. 2007. “Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology.” Nat. Methods 4 (3): 257-262. (hereinafter referred to as “SMart Amplification Process methods”).
In the isothermal amplification method of the present invention, a second primer may be used in combination with the first primer shown in the (X). It is preferable that the first primer and the second primer are a pair of primers. Examples of the pair of primers include an asymmetric pair in which the first primer and the second primer are different from each other in morphology and a symmetric pair in which the first primer and the second primer are identical to each other in morphology. Hereinafter, a primer set including the asymmetric pair of primers also is referred to as an asymmetric primer set, and a primer set including the symmetric pair of primers also is referred to as a symmetric primer set. The asymmetric primer set is suitable for, for example, the SMart Amplification Process method, and the symmetric primer set is suitable for, for example, the LAMP method.
The isothermal amplification method of the present invention will be described with reference to an example where the method is carried out using the above-described asymmetric primer set and an example where the method is carried out using the above-described symmetric primer set. It is to be noted, however, the present invention is not limited thereto.
Among isothermal amplification methods, the SMart Amplification Process method can amplify a target nucleic acid sequence with excellent specificity, for example. Accordingly, the SMart Amplification Process method makes it possible to judge, for example, the presence or absence of a mutation in a gene, particularly the presence or absence of single nucleotide mutation or the presence or absence of base deletion or base insertion by gene amplification.
As described above, the asymmetric primer set is a primer set having an asymmetric pair of primers composed of the first primer and the second primer that are different from each other in morphology. Particularly, it is preferable that the asymmetric primer set is used for the SMart Amplification Process method. Hereinafter, this primer set also is referred to as a “SMart Amplification Process primer set”.
In the action mechanism described above, the phenomenon in which the sequence (B′) hybridizes to the sequence (Bc) typically occurs due to the presence of the complementary regions on the same strand. Generally, when a double-stranded nucleic acid is dissociated into a single strand, partial dissociation starts from the ends thereof or from the relatively unstable portions other than the ends. In the double-stranded nucleic acid produced through the extension reaction caused by the above-mentioned first primer, base pairs located in the end portion are in a state of equilibrium between dissociation and binding at relatively high temperatures and thereby a double strand is retained as a whole. In such a state, when a sequence complementary to the dissociated portion located at the end is present on the same strand, a stem-loop structure can be formed in a metastable state. This stem-loop structure does not exist stably. However, another identical primer binds to the complementary strand portion (the sequence (A) on the template nucleic acid) exposed due to the formation of the stem-loop structure, and thereby a polymerase causes the extension reaction immediately. Accordingly, while the strand synthesized previously is displaced and thereby is released, a new double-stranded nucleic acid can be produced at the same time.
The design criteria for the first primer according to a preferred aspect of the present invention are as follows, for example. First, in order for a new primer to anneal to the template nucleic acid efficiently after a complementary strand to the template nucleic acid is synthesized through the extension of a preceding primer, it is necessary to allow the sequence (A) portion located on the template nucleic acid to be a single strand through the formation of the stem-loop structure at the 5′ end of the complementary strand synthesized as described above. For that purpose, a ratio of (X−Y)/X is important. That is a ratio of the difference (X−Y) to the number X, wherein X denotes the number of bases contained in the sequence (Ac′) and Y indicates the number of bases contained in the region flanked by the sequence (A) and the sequence (B) in the target nucleic acid sequence. However, the portion that is present on the 5′ side with respect to the sequence (A) on the template nucleic acid and that is not associated with the hybridization of the primer is not required to be a single strand. Furthermore, in order for a new primer to anneal to the template nucleic acid efficiently, it is also necessary that the above-mentioned stem-loop structure is formed efficiently. For the efficient formation of the stem-loop structure, i.e. for efficient hybridization between the sequence (B′) and the sequence (Bc), the distance (X+Y) between the sequence (B′) and the sequence (Bc) is important. Generally, the optimal temperature for the primer extension reaction is a maximum of around 72° C. It is difficult to dissociate the extended strand over a long region at such low temperatures. Therefore, conceivably, in order for the sequence (B′) to hybridize to the sequence (Bc) efficiently, it is preferable that a smaller number of bases exist between the two sequences. On the other hand, conceivably, in order for the sequence (B′) to hybridize to the sequence (Bc) to allow the sequence (A) portion located on the template nucleic acid to be a single strand, it is preferable that a larger number of bases exist in the sequence (B′).
From such factors as described above, the aforementioned first primer according to a preferred embodiment of the present invention is designed so that (X−Y)/X is, for example, at least −1.00, preferably at least 0.00, more preferably at least 0.05, and still more preferably at least 0.10 but is, for example, 1.00 or lower, preferably 0.75 or lower, more preferably 0.50 or lower, and still more preferably 0.25 or lower, in the case where no intervening sequence is present between the sequence (Ac′) and the sequence (B′) that compose the primer. Moreover, (X+Y) is preferably at least 15, more preferably at least 20, and still more preferably at least 30 but is preferably 50 or less, more preferably 48 or less, and still more preferably 42 or less. When an intervening sequence (the number of bases contained therein is Y′) is present between the sequence (Ac′) and the sequence (B′) that compose the primer, the first primer according to the preferred embodiment of the present invention is designed so that, for example, {X−(Y−Y′)}/X is at least −1.00, preferably at least 0.00, more preferably at least 0.05, and still more preferably at least 0.10 but is, for example, 1.00 or lower, preferably 0.75 or lower, more preferably 0.50 or lower, and still more preferably 0.25 or lower. Moreover, (X+Y+Y′) is, for example, at least 15, preferably at least 20, and more preferably at least 30 but is, for example, 100 or less, preferably 75 or less, and more preferably 50 or less.
Preferably, the aforementioned first primer has a strand length that enables base pairing with the target nucleic acid while allowing the necessary specificity to be maintained under given conditions, for example. The strand length of this primer is preferably 15 to 100 nucleotides and more preferably 20 to 60 nucleotides. The lengths of the sequence (Ac′) and the sequence (B′) that compose the first primer each are preferably 5 to 50 nucleotides and more preferably 7 to 30 nucleotides. Furthermore, an intervening sequence that does not affect the reaction may be inserted between the sequence (Ac′) and the sequence (B′) if necessary.
As described above, the second primer included in the primer set according to the present invention contains, for example, in its 3′ end portion, a sequence (Cc′) that hybridizes to a sequence (C) located in the 3′ end portion of a complementary sequence (the strand located on the opposite side to the strand to which the first primer hybridizes) to the target nucleic acid sequence. The second primer also contains, on the 5′ side of the sequence (Cc′), a folded sequence (D-Dc′) that contains, on the same strand, two nucleic acid sequences that hybridize to each other. Such a second primer has a structure like the one shown in
An example of the conceivable action mechanism of the isothermal amplification reaction that is caused by the above-mentioned first primer and second primer is described with reference to
The SMart Amplification Process primer set may include a third primer in addition to the first primer and the second primer. The third primer hybridizes to, for example, the target nucleic acid sequence or the complementary sequence thereto. However, the third primer does not compete with other primers for hybridization to the target nucleic acid sequence or the complementary sequence thereto. In the present invention, the expression “does not compete” means that hybridization of the third primer to a target nucleic acid does not hinder other primers from providing origins for complementary strand synthesis.
When the target nucleic acid sequence has been amplified with the first primer and the second primer, the amplification product contains the target nucleic acid sequence and the complementary sequence thereto alternately as described above. The amplification product has, at its 3′ end, a folded sequence or a loop structure. It provides the origin of complementary strand synthesis and thereby extension reactions occur consecutively therefrom. It is preferable that when such an amplification product becomes a single strand partially, the third primer can anneal to the target sequence that is present in the single strand portion. This allows the target nucleic acid sequence contained in the amplification product to be provided with a new origin of complementary strand synthesis. Then an extension reaction occurs therefrom. Thus the isothermal amplification reaction is performed much quicker.
The third primer is not limited and may be of one kind, or for example, in order to improve the speed and specificity of the isothermal amplification reaction, at least two kinds of third primers may be used simultaneously. Typically, such third primers have, for example, different sequences from those of the first primer and the second primer. However, each of the third primers may hybridize to a region, a part of which is hybridized by the first or second primer, as long as they do not compete with the first or second primer. The strand length of the third primer is preferably 2 to 100 nucleotides, more preferably 5 to 50 nucleotides, and further preferably 7 to 30 nucleotides.
The third primer is intended mainly to provide an auxiliary function to advance the isothermal amplification reaction caused by the first primer and the second primer much quicker. Hence, it is preferable that the third primer have a lower Tm than that of each 3′ end of the first primer and the second primer. Furthermore, it is preferable that the amount of the third primer to be added to the amplification reaction solution is smaller than that of each of the first primer and the second primer to be added thereto, for example.
The above-described third primer can be one that allows an origin of complementary strand synthesis to be provided for a loop portion, with a template having a structure capable of forming the loop, as described in, for example, WO 02/24902. The third primer, however, is not limited thereto. That is, it can be any primer that provides an origin of complementary strand synthesis for any site as long as the site is within the target nucleic acid sequence, for example.
In the SMart Amplification Process primer set, for example, either one of the first primer or the second primer or both of them may be a labeled nucleic acid(s) labeled with, for example, a fluorescent dye or the like, or the third primer may be the labeled nucleic acid. Either the first primer or the second primer or both of them and the third primer may be the labeled nucleic acids.
When the isothermal amplification method of the present invention is used, for example, for the mutation detection method to be described later, it is preferable that the SMart Amplification Process primer set is designed as follows. That is, preferably, the SMart Amplification Process primer set is designed so that a nucleic acid sequence having a mutation at a target site (hereinafter referred to as a “mutated nucleic acid sequence”) or a nucleic acid sequence having no mutation at the target site (hereinafter referred to as a “wild-type nucleic acid sequence”) is a target nucleic acid sequence, and the site where the target mutation occurs is contained in the sequence (A), the sequence (B), or the sequence (C), or is located between the sequence (A) and the sequence (B) or between the sequence (A) and the sequence (C).
When using a primer set designed so that a nucleic acid sequence containing a mutation at the target site (mutated sequence) is a target nucleic acid sequence, for example, the presence of an amplification product after the isothermal amplification reaction indicates the presence of the mutated sequence, while the absence of or reduction in the amplification product indicates the absence of the mutated sequence. On the other hand, when using a primer set designed so that a nucleic acid sequence containing no mutation at the target site (wild-type sequence) is a target nucleic acid sequence, for example, the presence of an amplification product after the isothermal amplification reaction indicates the absence of the mutated sequence, while the absence of or reduction in the amplification product indicates the presence of the mutated sequence. The expression “reduction in the amplification product” means a reduction in the amount of the amplification product obtained as compared to the amount of the amplification product that is obtained when the target nucleic acid sequence is present in the nucleic acid sample.
Preferably, the primer set is designed so that the target site is contained in the sequence (A). In the case of using such a primer set, for example, when the target nucleic acid sequence (e.g., a wild-type sequence) is contained in the nucleic acid sample, the first primer anneals to the sequence (A) in the isothermal amplification reaction and thereby an amplification product is obtained. On the other hand, when a nucleic acid sequence (e.g., a mutated sequence) that is different from the target nucleic acid sequence is contained in the nucleic acid sample, it is difficult for the first primer to anneal to the sequence (A) in the isothermal amplification reaction. Accordingly, in this case, no amplification product is obtained or a considerably reduced amount of amplification product is obtained. Preferably, the sequence (Ac′) contained in the first primer is a sequence that is complementary to the sequence (A).
Preferably, the primer set is one designed so that, for example, the target site is contained in the sequence (C). In the case of using such a primer set, for example, when the target nucleic acid sequence (e.g., a wild-type sequence) is contained in the nucleic acid sample, the second primer anneals to the sequence (C) in the isothermal amplification reaction and thereby an amplification product is obtained. On the other hand, when a nucleic acid sequence (e.g., a mutated sequence) that is different from the target nucleic acid sequence is contained in the nucleic acid sample, it is difficult for the second primer to anneal to the sequence (C) in the isothermal amplification reaction. Therefore, in this case, no amplification product is obtained or a considerably reduced amount of amplification product is obtained. Preferably, the sequence (Cc′) contained in the second primer is a sequence that is complementary to the sequence (C).
Preferably, the primer set is one designed so that, for example, the target site is contained in the sequence (B). In the case of using such a primer set, for example, when the target nucleic acid sequence (e.g., a wild-type sequence) is contained in the nucleic acid sample, after the first primer anneals to the sequence (A) to cause an extension reaction, a sequence (B′) that is contained in the primer hybridizes to a sequence (Bc) located on the extended strand in the isothermal amplification reaction. Therefore a stem-loop structure is formed efficiently. This efficient formation of the stem-loop structure allows another first primer to anneal to the template. Accordingly, the action mechanism shown in
Preferably, the primer set is one designed so that, for example, the target site is located between the sequence (A) and the sequence (B). In the case of using such a primer set, when the target nucleic acid sequence (e.g., a wild-type sequence) is contained in the nucleic acid sample, after the first primer anneals to the sequence (A) to cause an extension reaction, the sequence (B′) that is contained in the primer hybridizes to the sequence (Bc) located on the extended strand and thereby a stem-loop structure is formed efficiently in the isothermal amplification reaction. This efficient formation of the stem-loop structure allows another first primer to anneal to the template. Accordingly, the action mechanism shown in
Preferably, the primer set is one designed so that the target site is located between the sequence (A) and the sequence (C). In the case of using such a primer set, when the target nucleic acid sequence is contained in the nucleic acid sample (e.g., a wild-type sequence), after the first primer anneals to the sequence (A) to cause an extension reaction, the sequence (B′) that is contained in the primer hybridizes to the sequence (Bc) located on the extended strand and thereby a stem-loop structure is formed efficiently in the isothermal amplification reaction. This efficient formation of the stem-loop structure allows another first primer to anneal to the template. Accordingly, the action mechanism shown in
As described above, the symmetric primer set includes a symmetric pair of primers composed of the first primer and the second primer that are identical to each other in morphology. Particularly, it is preferable that the primer set is used for the LAMP method. Hereinafter, this primer set also is referred to as a “LAMP primer set”.
In the case where the first primer is designed, for example, so as to anneal to an antisense strand, the second primer is designed so as to anneal to a sense strand. In the case where the first primer is designed, for example, so as to anneal to a sense strand, the second primer is designed so as to anneal to an antisense strand. When the first primer has annealed to, for example, a target nucleic acid sequence in an antisense strand, it is preferable that the second primer anneal to a region in a sense strand corresponding to a 5′ side region with respect to the site of the antisense strand to which the first primer has annealed.
In the LAMP method, for example, four kinds of primers are necessary. They recognize six regions, so that a target gene can be amplified. That is, in this method, for example, a first primer anneals to a template strand to cause an extension reaction first. Subsequently, the extended strand produced by the first primer separates from the template strand due to the strand displacement reaction caused by a second primer designed upstream from the first primer. At this time, a stem-loop structure is formed in the 5′ end portion of the extended strand due to the structure of the first-primer extension product that has been removed. Similar reactions occur in the other strand of the double-stranded nucleic acid or on the 3′ end side of the first-primer extension product that has been removed. These reactions are repeated and thereby the target nucleic acid is amplified. The template used in the LAMP method has, for example, at the 3′ end and the 5′ end on the same strand, regions composed of base sequences complementary to each other in the respective end regions. With this template (also referred to as a “dumbbell-type template nucleic acid”), loops are formed, in which base pairing can occur between the base sequences complementary to each other when they anneal to each other. The LAMP method can be performed according to, for example, WO 00/28082 or WO 01/034838.
The isothermal amplification kit of the present invention is a kit to be used in the isothermal amplification method of the present invention. The kit is characterized in that it includes the isothermal amplification DNA polymerase according to the present invention. According to the kit of the present invention, for example, the above-described isothermal amplification method of the present invention can be carried out easily and conveniently. It is only necessary that the isothermal amplification kit of the present invention include the DNA polymerase of the present invention, and configurations and the like other than this are not limited. The above-described other configurations and the like can be determined as appropriate depending on the above-described isothermal amplification method. The isothermal amplification kit of the present invention may include, for example, a primer or a primer set, and a user manual in addition to the DNA polymerase of the present invention. The primer and the primer set may be those described above. Furthermore, the isothermal amplification kit of the present invention may further include: for example, a substrate such as dNTP mix (dATP, dTTP, dCTP, and dGTP); a buffer such as a Tris-HCl buffer, a tricine buffer, a sodium phosphate buffer, or a potassium phosphate buffer; a catalyst such as magnesium chloride, magnesium acetate, or magnesium sulfate; an additive such as dimethyl sulfoxide (DMSO) or betaine (N,N,N-trimethylglycine); an acidic substance and a cation complex described in WO 99/54455; an enzyme stabilizer; etc. The enzyme stabilizer is not limited. Examples thereof include glycerol, bovine serum albumin, and saccharide. Particularly, the enzyme stabilizer is preferably saccharide, more preferably monosaccharide or oligosaccharide, and still more preferably trehalose, sorbitol, mannitol, or a mixture of two or more of them. The isothermal amplification kit of the present invention further may include a melting temperature regulator. Examples of the melting temperature regulator include DMSO, betaine, formamide, glycerol, and arbitrary combinations thereof. It preferably is DMSO. Furthermore, when the nucleic acid sample used for the isothermal amplification method contains RNA and the RNA is used as a template, it is preferable that the kit further contain a reverse transcriptase, for example. In the isothermal amplification kit of the present invention, the ratio of these reagents, etc. are not limited and can be determined as appropriate by those skilled in the art.
The isothermal amplification kit of the present invention preferably further includes the above-described mismatch binding protein, and it preferably includes the single-stranded binding protein (SSB) in addition to the mismatch binding protein.
In the isothermal amplification kit of the present invention, reagents such as the DNA polymerase and the primer may be contained separately in different containers or may be contained in the same container. The isothermal amplification kit of the present invention also can be referred to as, for example, an isothermal amplification reagent to be used in the isothermal amplification method of the present invention.
In microorganisms of the genus Bacillus as the related genus, generally, a phoR gene is conserved upstream and a mutM gene is conserved downstream of a gene encoding DNA polymerase I (polA gene). Thus, a DNA polymerase gene (polA) of, for example, the genus Alicyclobacillus can be isolated by, for example, determining the base sequences of the conserved genes, designing a polA cloning primer based on the base sequences thus determined, and carrying out gene amplification. A specific example will be given below. It is to be noted, however, the present invention is not limited thereto.
First, from the conserved regions of the phoR gene and the mutM gene of a microorganism of the genus Bacillus, a primer or one or more pairs of primers for amplifying these genes are designed. Using such a primer set, each of the genes is amplified with a genomic DNA of a target bacterium (e.g., a microorganism of the genus Alicyclobacillus) being a template. Then, the base sequences of the resultant amplification products are determined.
Owing to the genomic structures of various microorganisms of the genus Bacillus, the phoR gene, the polA gene, and the mutM gene are transcribed in the same direction. Thus, based on the information as to the thus-determined base sequences, a primer (forward primer) homologous to a sense strand (complementary to an antisense strand) of the phoR gene and a primer (reverse primer) homologous to an antisense strand (complementary to a sense strand) of the mutM gene are designed as specific primers for polA cloning. Then, using these primers, DNA containing the polA gene is amplified with the genomic DNA of the bacterium being a template. By cloning the resultant amplification product, it is possible to obtain DNA containing the polA gene derived from the target bacterium. The length of the primer is not limited, and can be adjusted as appropriate depending on various experimental conditions. It is, for example, 15 to 50 mer, preferably 18 to 40 mer, and most preferably 25 to 35 mer.
A method for isolating a DNA polymerase gene from a bacterium belonging to or related to the genus Alicyclobacillus using such primers may include, for example, the following steps (A) to (D). The method may further include the following step (E).
(A) amplifying a DNA fragment of a phoR gene using a primer having a base sequence represented by SEQ ID NO. 1 or 2 and a primer having a base sequence represented by any of SEQ ID NOs. 3 to 5 with a genomic DNA extracted from the bacterium being a template
(B) amplifying a DNA fragment of a mutM gene using a primer having a base sequence represented by any of SEQ ID NOs. 6 to 8 and a primer having a base sequence represented by any of SEQ ID NOs. 9 to 11 with the genomic DNA extracted from the bacterium being a template
(C) determining a base sequence of each of the DNA fragments amplified in the steps (A) and (B)
(D) amplifying a DNA fragment containing a polA gene using a primer having a partial sequence of a sense strand of the phoR gene and a primer having a partial sequence of an antisense strand of the mutM gene, which are designed based on the base sequences determined in the step (C), with the genomic DNA extracted from the bacterium being a template
(E) cloning the DNA fragment amplified in the step (D)
The length of the primer is not limited, and can be adjusted as appropriate depending on various experimental conditions. It is, for example, 15 to 50 mer, preferably 18 to 40 mer, and most preferably 25 to 35 mer. In the step (A), the primer having the base sequence represented by SEQ ID NO. 1 or 2 is a forward primer, and the primer having the base sequence represented by any of SEQ ID NOs. 3 to 5 is a reverse primer. In the step (B), the primer having the base sequence represented by any of SEQ ID NOs. 6 to 8 is a forward primer, and the primer having the base sequence represented by any of SEQ ID NOs. 9 to 11 is a reverse primer.
As the primer set in the step (D), for example, it is possible to use a forward primer having a base sequence represented by any of SEQ ID NOs. 12 to 17 and a reverse primer having a base sequence represented by SEQ ID NO. 18 in combination as will be described later. Furthermore, as a primer set for amplifying a full-length translation region, it is possible to use, for example, the combination of a forward primer having a base sequence represented by SEQ ID NO. 19 and a reverse primer having a base sequence represented by SEQ ID NO. 21 as will be described later. Still further, as a primer set for amplifying a coding sequence of a DNA polymerase in which an N-terminal region is deleted, it is possible to use, for example, the combination of a forward primer having a base sequence represented by SEQ ID NO. 20 and a reverse primer having a base sequence represented by SEQ ID NO. 21 as will be described later.
Examples of the DNA encoding the isothermal amplification DNA polymerase according to the present invention include DNAs shown in the following items (a′) to (d′), which encode the DNA polymerases shown in the above-described items (a) to (d), respectively.
(a′) DNA having a base sequence represented by SEQ ID NO. 22
(b′) DNA having a base sequence represented by SEQ ID NO. 24
(c′) DNA having a base sequence obtained by deletion of any number from 1 to 334 of consecutive codons starting from the 5′ end in the base sequence represented by SEQ ID NO. 22
(d′) DNA that has a base sequence obtained by deletion, substitution, insertion, or addition of one or more bases in the base sequence described in any of the items (a′) to (c′) and encodes a protein having a DNA polymerase activity
The base sequence represented by SEQ ID NO. 22 is, for example, a DNA sequence encoding the amino acid sequence represented by SEQ ID NO. 23. The base sequence represented by SEQ ID NO. 24 is, for example, a DNA sequence encoding the N-terminal deleted DNA polymerase represented by SEQ ID NO. 25. Note here that the present invention is not limited to these sequences.
Furthermore, DNA used in the present invention may be, for example, a DNA shown in the following (e′), which encodes a DNA polymerase shown in the above-described item (e), as long as the DNA polymerase encoded thereby has a DNA polymerase activity.
(e′) DNA that has a base sequence with a homology of, for example, at least 80% to the base sequence represented by any of the items (a′) to (c′) and encodes a protein having a DNA polymerase activity.
The homology can be calculated using the BLAST or the like under default conditions, for example. The homology is, for example, at least 80% or more, preferably 90% or more, and still more preferably 95% or more. The present invention also includes DNA that has a base sequence with a homology of, for example, at least 80% or more, preferably 90% or more, and more preferably 95% or more to DNA having the base sequence represented by SEQ ID NO. 24 when the homology is calculated using the BLAST or the like under the default conditions and encodes a protein having the DNA polymerase activity but lacking the 5′→3′ exonuclease activity. Furthermore, the present invention also includes RNA to the above-described DNA, e.g., mRNA transcribed from the above-described DNA, or an antisense RNA.
Furthermore, DNA in the present invention may be DNA shown in the following item (f′), for example, as long as a polymerase encoded thereby has a DNA polymerase activity.
(f′) DNA that hybridizes to DNA having the base sequence described in any of the items (a′) to (c′) under stringent conditions and encodes a protein having a DNA polymerase activity
In the item (f′), “hybridization under stringent conditions” means hybridization under experimental conditions well known to those skilled in the art, for example. Specifically, the term “stringent conditions” refers to conditions such that a hybrid formed is identified after carrying out hybridization at 60° C. to 68° C. in the presence of 0.7 to 1 mol/l NaCl and then carrying out washing at 65° C. to 68° C. using a 0.1- to 2-fold SSC solution. Note here that 1×SSC is composed of 150 mmol/l NaCl and 15 mmol/l sodium citrate, for example. In order to select the stringency, for example, the salt concentration and the temperature in the washing step can be optimized as appropriate. Furthermore, it is common general technical knowledge in the art to add, for example, formamide, SDS, or the like to improve the stringency.
Furthermore, DNA in the present invention includes, for example, a degenerate variant thereof having the base sequence represented by SEQ ID NO. 22 or 24. Still further, the present invention includes DNA having a complementary sequence to the above-described DNA.
Although a method for introducing mutation in a gene is not limited, the introduction of mutation can be carried out by, for example, a known method such as the Kunkel method or the gapped duplex method, or a method equivalent thereto. Also, it is possible to use a mutation introducing kit utilizing site-directed mutagenesis (e.g., Mutant-K (TAKARA), Mutant-G (TAKARA), or the like), LA PCR in vitro Mutagenesis series kit (TAKARA), or the like, for example.
Next, a primer and a probe used for isolating a DNA polymerase from a living organism will be described. The primer and the probe each are, for example, a fragment of any of the above-described DNA of the present invention, and the number of bases is, for example, 5 to 50. The lower limit of the number of bases is, for example, 5 or more, preferably 10 or more, and more preferably 15 or more, and the upper limit of the number of bases is, for example, 50 or less, preferably 30 or less, and more preferably 25 or less. Furthermore, a specific examples of a preferable range of the number of bases is, for example, from 10 to 50, preferably from 10 to 30, and more preferably from 15 to 25. The length of a base sequence to be amplified is not particularly limited. Specific examples of the primer of the present invention include those represented by SEQ ID NOs. 1 to 21. One of them may be used alone, or two or more of them may be used in combination. In particular, it is preferable that at least one forward primer selected from the primers represented by SEQ ID NOs. 12 to 17 and a reverse primer that is the primer represented by SEQ ID NO. 18 are used in combination. When isolating a full-length translation region (ORF) of the polA gene, it is preferable that, for example, a forward primer represented by SEQ ID NO. 19 and a reverse primer represented by SEQ ID NO. 21 are used in combination. Furthermore, when isolating a region corresponding to Escherichia coli Klenow fragment in the polA gene, it is preferable that, for example, a forward primer represented by SEQ ID NO. 20 and a reverse primer represented by SEQ ID NO. 21 are used in combination.
Next, a recombinant vector that can be used for expression of the isothermal amplification DNA polymerase according to the present invention will be described. The recombinant vector is characterized in that it contains any of the above-described DNAs of the present invention. The recombinant vector can be obtained by, for example, ligating (inserting) the DNA of the present invention to a suitable vector. The vector to which the DNA is inserted is not particularly limited, as long as, for example, it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA. Examples of the plasmid DNA include plasmids derived from Escherichia coli pBR322, pBR325, pUC118, pUC119, and the like), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, and the like), and plasmids derived from yeasts (e.g., YEp13, YEp24, YCp50, and the like). Examples of the phage DNA include λ phage (e.g., Charon 4A, Charon 21A, EMBL3, EMBL4, λgt10, λgt11, μZAP, and the like). Also, it is possible to use an animal virus such as retrovirus or vaccinia virus, an insect virus vector such as baculovirus, or the like.
A method for inserting the DNA into a vector is not particularly limited, and a conventionally known method can be employed. Specific examples include a method in which, for example, a purified DNA first is cleaved with a suitable restriction enzyme and the resultant DNA fragment is inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, thus ligating the DNA fragment to the vector. It is preferable that the DNA of the present invention is incorporated in the vector so as to allow the expression of a protein encoded by the DNA, for example. Thus, to the vector used in the present invention, for example, not only a promoter (e.g., a trp promoter, a lac promoter, a PL promoter, a tac promoter, or the like) but also a cis element such as an enhancer, a splicing signal, a poly-A addition signal, a selection marker, a ribosome binding sequence (an SD sequence, a KOZAK sequence, or the like), and the like may be ligated, if desired. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, and a neomycin resistance gene.
A transformant that can be used for the production of an isothermal amplification DNA polymerase according to the present invention will be described. The transformant is characterized in that it contains the above-described recombinant vector. The transformant is obtained by, for example, transfecting the above described DNA into a host so that the isothermal amplification DNA polymerase according to the present invention can be expressed. Specifically, it is preferable to transfect the recombinant vector of the present invention. In many cases, a vector is used for transformation because it allows the transformation to be carried out easily and efficiently, for example. The host is not particularly limited as long as it can express the protein of the present invention. Examples of the host include bacteria or the like belonging to: the genus Escherichia such as Escherichia coli; the genus Bacillus such as Bacillus subtilis; the genus Pseudomonas such as Pseudomonas putida; and the genus Rhizobium such as Rhizobium meliloti. In addition, yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells such as a COS cell and CHO cell also can be used. Alternatively, it is also possible to use an insect cell such as Sf-9 and Sf-21, for example.
A method for carrying out the transformation is not limited, and a conventionally known method can be employed. Specific examples include a method using calcium ions (Cohen, S. N. et al. (1972) Proc. Natl. Acad. Sci., USA 69, 2110-2114), a DEAE dextran method, and an electroporation method.
The isothermal amplification DNA polymerase according to the present invention can be produced by, for example, culturing the transformant and collecting a protein (DNA polymerase) from the resultant culture. The term “culture” means, for example, not only culture supernatant but also cultured cells or cultured bacterial cells and a homogenate thereof. Furthermore, “a method for culturing the transformant of the present invention” is carried out in accordance with, for example, a usual method applied to the culture of the host, and the conditions etc. thereof can be determined as appropriate depending on the kind of the host, etc., for example.
After the culture, in the case where the protein (DNA polymerase) of the present invention is produced in the bacterial cells or the cells, the protein is extracted by, for example, homogenizing the bacterial cells or the cells. On the other hand, in the case where the protein (DNA polymerase) of the present invention is produced outside the bacterial cells or the cells, for example, the culture solution is used as it is, or the bacterial cells or the cells are removed from the culture solution by centrifugation or the like. Thereafter, the protein (DNA polymerase) of the present invention can be purified from the culture by using a biochemical method commonly used for isolation and purification of proteins either alone or, if necessary, in combination with another method. Examples of the method for conducting the isolation and purification include ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography. Furthermore, for example, in the case where a protein to be expressed has a tag sequence for purification, the tag sequence can be removed by a protease treatment or the like during or after the purifying step.
Furthermore, an antibody against the DNA polymerase of the present invention will be described. The term “antibody” encompasses, for example, any suitable fragments and derivatives. Examples of the antibody in such a broad sense include polyclonal antibodies, monoclonal antibodies, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, diabody (two copies of the same Fv fragment, fused to each other), single-chain antibodies, and multi-specific antibodies each composed of two or more antibody fragments. The antibody can be used for, for example, purification of the isothermal amplification DNA polymerase according to the present invention. The method for producing the antibody is not limited, and the antibody can be produced by a conventionally known method, for example.
In the present invention, for example, general methods based on molecular biology, microbiology, a recombinant DNA technology, and the like can be conducted with reference to standard reference books in the art. Examples of these books include: Molecular Cloning: A Laboratory Manual, Third Edition (Sambrook & Russell, Cold Spring Harbor Laboratory Press, 2001); Current Protocols in Molecular Biology (edited by Ausubel et al., John Wiley & Sons, 1987); a series of Methods in Enzymology (Academic Press); PCR Protocols: Methods in Molecular Biology (edited by Bartlett & Stirling, Humana Press, 2003); and Antibodies: A Laboratory Manual (edited by Harlow & Lane, Cold Spring Harbor Laboratory Press, 1987). Furthermore, reagents, kits, etc. referred to herein are available from commercial vendors such as, for example, Sigma, Aldrich, Invitrogen/GIBCO, Clontech, Stratagene, Qiagen, Promega, Roche Diagnostics, Becton-Dickinson, and Takara Bio Inc.
The present invention will be described in further detail with reference to the following examples. It is to be noted, however, the present invention is by no means limited to these examples.
From cultured bacterial cells of A. acidocaldarius subsp. Acidocaldarius JCM 5260, genomic DNA was prepared according to a conventional method. Based on the fact that phoR is conserved upstream of polA and mutM is conserved downstream of the polA in polA gene structures of other species of the genus Bacillus, primers shown below were synthesized from the conserved regions of the respective genes.
In the above sequences, R denotes a purine base of guanine or adenine, Y denotes a pyrimidine base of thymine or cytosine, M denotes adenine or cytosine, K denotes guanine or thymine, S denotes guanine or cytosine, W denotes adenine or thymine, D denotes adenine, guanine, or thymine, H denotes adenine, cytosine, or thymine, V denotes adenine, guanine, or cytosine, N denotes any one of four kinds of bases (guanine, adenine, cytosine, thymine). The number in parentheses denotes the number of the kinds of each primer obtained through combination. Furthermore, Fwd denotes a forward primer, and Rev denotes a reverse primer. The same applies to sequences described below.
Using the thus-prepared genomic DNA as a template, DNA amplification was carried out by PCR with the use of each combination of a forward (Fwd) primer and a reverse (Rev) primer. The amplified DNA fragment was cloned into Plasmid pGEM-T (trade name, Promega KK). Specifically, the DNA fragment of the phoR gene was amplified using either one of phoRFwd1 and phoRFwd2 as a forward primer and any one of phoRRev1, phoRRev2, and phoRRev3 as reverse primer. Furthermore, the DNA fragment of the mutM gene also was amplified using any one of mutMRev1, mutMRev2, and mutMRev3 as a forward primer and any one of mutMRev1, mutMRev2, and mutMRev3 as a reverse primer. Then, each of the thus-obtained DNA fragments was cloned into the plasmid, and the base sequence inside thereof was determined.
Based on the base sequences thus determined, the following primers for cloning a polA gene were synthesized.
Using the prepared genomic DNA as a template, DNA amplification was carried out by PCR with the use of the combination of any one of the Aac cloning F1 to F6 as a forward primer and the Aac cloning R1 as a reverse primer. The amplified DNA fragment was cloned into Plasmid pGEM-T (trade name, Promega KK). Then, the base sequence of the amplified DNA fragment cloned into the plasmid was determined. Subsequently, based on the thus-determined base sequence and the base sequence presumed to be the gene (polA) of Alicyclobacillus acidocaldarius DNA polymerase (Aac DNA polymerase I), the following primer pairs were designed. Using these primer pairs, a full-length translation region (ORF) moiety was amplified by PCR.
The resultant PCR amplification product and a plasmid pYSN were digested with restriction enzymes NdeI and KpnI and were mixed together, whereby the PCR amplification product was ligated into the plasmid pYSN. Thus, an Aac DNA polymerase expression plasmid pAac was constructed.
Furthermore, from the thus-obtained Aac DNA polymerase expression plasmid pAac, the base sequence of the polA gene was determined. Then, a DNA fragment corresponding to the N-terminal of Escherichia coli Klenow fragment was amplified by PCR using the following primer pair.
The resultant PCR amplification product and a plasmid pYSN were digested with restriction enzymes NdeI and KpnI and mixed together, whereby the PCR amplification product was ligated into the plasmid pYSN. Thus, expression plasmid pdNAac for N-terminal deleted Aac DNA polymerase (hereinafter also referred to as “ΔN Aac DNA polymerase”, “Aac DNA polymerase large fragment” or “Aac DNA polymerase I”) was constructed.
Escherichia coliXL1-Blue having pAac or pdNAac was cultured in 5 ml of LB medium containing 100 μg/ml ampicillin at 37° C. overnight. The resultant solution was used as a preculture solution. 5 ml of this preculture solution was inoculated into 500 ml of LB medium containing 100 μg/ml ampicillin, and was subjected to shaking culture at 37° C. and 200 rpm (Orbital Shaking Incubator, FIRSTEK OSI-502LD). At the time when the OD 600 nm of the culture solution reached around 0.5, IPTG was added so that the final concentration thereof was 1 mmol/l. The resultant mixture was subjected to further shaking culture at 37° C. and 200 rpm for 1 to 2 hours. Subsequently, the culture solution was transferred into a centrifuge tube and subjected to centrifugation at 4,000×g for 10 minutes. As a result, a precipitate was obtained. This precipitate was suspended in 30 ml of 1×PBS, and the resultant suspension again was centrifuged at 4,000×g for 10 minutes, thereby washing bacterial cells. The resultant precipitate was suspended in 25 ml of 1×PBS, and sonication of the cells for 10 seconds was carried out six times in total (MISONIX Astrason Ultrasonic processor XL). The sample after being subjected to the sonication was centrifuged at 15,000×g for 30 minutes. Thus, a supernatant was obtained. To this supernatant, a 30% polyethyleneimine solution was added so that the final concentration thereof was 0.1%, and they were mixed together. The resultant mixture was allowed to stand in ice for 30 minutes and then was centrifuged at 15,000×g for 30 minutes. Thus, a supernatant was obtained. The thus-obtained supernatant was used as a crude liquid extract.
Ion exchange chromatography was performed using AKTA Prime high performance liquid chromatography system manufactured by GE Healthcare and an anion-exchange column HiTrapQ manufactured by GE Healthcare. As a running buffer, 50 mmol/l Tris-HCl (pH 7.6) containing 10 mmol/1 2-mercaptoethanol was used. The column was equilibrated at a flow rate of 1 ml/min, and the above-described crude liquid extract was applied thereto. Thereafter, a non-adsorbed fraction was washed with the running buffer. An adsorbed fraction was eluted with a concentration gradient (from 0 to 1 mol/l) of about 15 column volumes of sodium chloride. The eluted fraction was fractionated every 1 ml, and each fraction was applied to SDS-PAGE to check a protein band. Then, the fraction having a protein band of the corresponding molecular weight was collected. The collected fraction was concentrated and desalted using an ultrafilter membrane, and this was used as an anion-exchange fraction.
Heparin affinity column chromatography was performed using AKTA Prime high performance liquid chromatography system manufactured by GE Healthcare and a heparin affinity column HiTrap Heparin manufactured by GE Healthcare. As a running buffer, the same solution as in the above-described anion-exchange column chromatography was used. The column was equilibrated at a flow rate of 1 ml/min, and the above-described anion-exchange fraction was applied thereto. Thereafter, a non-adsorbed fraction was washed with the running buffer. An adsorbed fraction was eluted with a concentration gradient (from 0 to 1 mol/l) of about 22 column volumes of sodium chloride. The eluted fraction was fractionated every 1 ml, and each fraction was applied to SDS-PAGE to check a protein band. Then, the fraction having a protein band of the corresponding molecular weight was collected. Using an ultrafilter membrane, the collected fraction then was subjected to buffer exchange with 50 mmol/l Tris-HCl (pH 8.0) containing 0.2 mol/l sodium chloride and further to concentration. This was used as a heparin fraction.
Gel filtration column chromatography was performed using AKTA 10XT high performance liquid chromatography system manufactured by GE
Healthcare and a gel filtration column HiLoad 16/60 Superdex 200 prep grade manufactured by GE Healthcare. As a running buffer, 50 mmol/l Tris-HCl (pH 8.0) containing 0.2 mol/l sodium chloride was used. The column was equilibrated at a flow rate of 1 ml/min, and the above-described heparin fraction was applied thereto. Thereafter, elution was performed using the running buffer. The eluted fraction was fractionated every 1 ml, and each fraction was applied to SDS-PAGE to check a protein band. Then, the fraction having a protein band of the corresponding molecular weight was collected. The collected fraction was concentrated using an ultrafilter membrane, and buffer exchange with a storage buffer was performed. This was used as a purified enzyme preparation. The composition of the storage buffer is: 50 mmol/l potassium chloride, 10 mmol/l Tris-HCl (pH 7.5), 1 mmol/l DTT, 0.1 mmol/l EDTA, 0.1% Triton X-100, and 50% glycerol.
A DNA polymerase activity of the purified enzyme preparation (Aac DNA polymerase large fragment) was measured using Picogreen dsDNA quantification reagent manufactured by Invitrogen with reference to Seville M. et al., BioTechniques Vol. 21, pp. 664-668 (1996). Specifically, the Picogreen dsDNA quantification reagent and a TE buffer were mixed together at a volume ratio of 1:345. Then, 173 μl of the resultant mixture was added to 27 μl of a mixture of M13 mp18 single-stranded DNA, a primer, dNTP, and the purified enzyme preparation. This reaction solution was left to stand at room temperature (37° C.) for 5 minutes. Thereafter, the fluorescence thereof was measured at an excitation wavelength of 480 nm and a measurement wavelength of 520 nm. At this time, the fluorescence of a commercially available Klenow fragment (commercially available Bst DNA polymerase; available from NEB) with a known unit (unit: an amount of enzyme required for incorporating 10 nmol of dNTP at 65° C. in 30 minutes) also was measured in the same manner. From the measured value thereof, an enzyme unit as a relative value was calculated. One example of the measurement is shown below. A standard curve was prepared for each measurement, and the reaction temperature was set to 37° C. Using the commercially available Bst DNA polymerase (available from NEB, hereinafter the same) as the standard, the fluorescence intensity was measured with respect to each dilution factor using the Picogreen dsDNA quantification reagent. The results thereof are shown in Table 1.
These results were plotted and regressed to a first-order linear equation. As a result, the following equation was obtained. The thus-obtained regression curve is shown in
y=8.3717x+55.292 (R2=0.9671)
Furthermore, the purified enzyme preparation (Aac DNA polymerase large fragment) subjected to the measurement exhibited an average fluorescence intensity of 98.03 (the first time: 97.789, the second time: 98.262) at the reaction temperature (room temperature: 37° C.). Thus, from the above-described regression linear equation, the DNA polymerase activity thereof was calculated to be about 5.10 units at the reaction temperature. From this result, it was confirmed that the purified enzyme preparation obtained in Example 2 was a DNA polymerase.
Activity measurement was carried out according to a method described in the above-described Non-Patent Document 2 (Notomi, T. et al., Nucleic Acids Research, 2000, Vol. 28, No. 12, e63). First, a mixture of each of synthetic DNAs (M13 mp 18 single-stranded DNA, 0.8 μmol/l FIP, 0.8 μmol/l BIP, 0.2 μmol/l F3, and 0.2 μmol/l B3), 1 mol/l betaine, 20 mmol/l Tris-HCl buffer (pH 8.8), 10 mmol/l potassium chloride, 10 mmol/l ammonium sulfate, 0.1% Triton X-100, and 2-4 mmol/l magnesium sulfate was prepared in an amount of 20 μl. The mixture was left to stand at 95° C. for 5 minutes and then on ice for 5 minutes. Then, 5 μl of the above-described purified enzyme preparation was added to the mixture, and the resultant mixture was left to stand at a predetermined reaction temperature (60° C. to 74° C.) for 1 hour. Thereafter, it was applied to agarose gel electrophoresis. At this time, Bst DNA polymerase (large fragment) with a known unit was applied to the electrophoresis in the same manner. Then, by comparing the band intensity of a purified product of the purified enzyme preparation after the electrophoresis and the band intensity of a purified product of the Bst DNA polymerase with a known unit after the electrophoresis, an enzyme unit as a relative value was calculated. Moreover, the electrophoresis also was carried out with respect to a blank obtained by adding the Tris-HCl buffer instead of the purified enzyme preparation (Aac DNA polymerase large fragment). The results thereof are shown in
Odd-numbered lanes (3, 5, 7, 9, 11, 13) in
Even-numbered lanes (2, 4, 6, 8, 10, 12) in
As can be seen from
According to the SMart Amplification Process method (the method proposed by Mitani et al. For example, see Patent Document 4 (Japanese Patent No. 3867926) and Patent Document 5), isothermal amplification of a nucleic acid was performed. 10 μl of a reaction solution having the following composition was prepared, and this was reacted at 65° C. for 60 minutes. This reaction was monitored in real time using Mx3000P (trade name, Stratagene). As a comparative example, the following reaction solution was prepared by adding a commercially available Bst DNA polymerase (large fragment) instead of the Aac DNA polymerase large fragment so that the final concentration thereof was 0.32 unit/μl, and the real time monitoring was conducted in the same manner. These results are shown in
As a SMart Amplification Process primer set, the following five kinds of primers were provided. A first primer is Turn-back Primer (TP), a second primer is Folding Primer (FP), and third primers are Boost Primer for wild-type detection (BPw), Outer Primer 1 (OP1), and Outer Primer 2 (OP2).
According to the SMart Amplification Process method using an Aac DNA polymerase large fragment, wild-type human ALDH2 was detected.
To 15 μl of blood containing hetero-type human ALDH2, 300 μl of 50 mmol/l NaOH was added. The resultant mixture was heat-treated at 98° C. for 3 minutes and then cooled with ice. This was used as a nucleic acid sample.
As a SMart Amplification Process primer set, the following five kinds of primers were provided. A first primer is Turn-back Primer (TP), a second primer is Folding Primer (FP), and third primers are Boost Primer for wild-type detection (BPw), Outer Primer 1 (OP1), and Outer Primer 2 (OP2).
(3) SMart Amplification Process reaction
A reaction solution having the following composition (composition per 25 μl) was prepared in the total amount of 75 and the reaction solution was reacted at 60° C. for 1 hour. During the reaction, the fluorescence intensity of the reaction solution was monitored in real time with Mx3000P (trade name, Stratagene) while maintaining an isothermal condition at 60° C. As a comparative example, the reaction solution was prepared by adding 6 U of a commercially available Bst DNA polymerase (large fragment) instead of the Aac DNA polymerase large fragment, and the real time monitoring was conducted in the same manner. 75 μl of the reaction solution was divided equally into three, and the reaction measurement was conducted with respect to each of 25 μl of the equally-divided reaction solutions. These results are shown in
As can be seen from
According to the LAMP method using an Aac DNA polymerase large fragment, wild-type human DIO2 was detected.
To 15 μl of blood containing hetero-type human DIO2, 30 μl of 50 mmol/l NaOH was added. The resultant mixture was heat-treated at 98° C. for 3 minutes and then cooled with ice. This was used as a nucleic acid sample.
As a LAMP primer sets, the following five kinds of primers were provided. TPw and TRFs2 form an asymmetric pair of primers.
A reaction solution having the following composition (composition per 25 μl) was prepared in the total amount of 75 and the reaction solution was reacted at 60° C. for 2 hours. During the reaction, the fluorescence intensity of the reaction solution was monitored in real time with Mx3000P (trade name, Stratagene) while maintaining an isothermal condition at 60° C. As a comparative example, the reaction solution was prepared by adding 6 U of a commercially available Bst DNA polymerase (large fragment) instead of the Aac DNA polymerase large fragment, and the real time monitoring was conducted in the same manner. 75 μl of the reaction solution was divided equally into three, and the reaction measurement was conducted with respect to each of 25 μl of the equally-divided reaction solutions. These results are shown in
As can be seen from
As specifically described above, the isothermal amplification DNA polymerase of the present invention can improve the amplification efficiencies of the specific isothermal amplification methods such as the SMart Amplification Process method and the LAMP method as compared with conventional DNA polymerases used in the isothermal amplification methods. Thus, the isothermal amplification DNA polymerase of the present invention allows nucleic acid amplification to be carried out in a shorter time than ever before. Moreover, since the time required for nucleic acid amplification can be shortened, for example, analysis of single nucleotide polymorphism (SNP) or the like utilizing the nucleic acid amplification can be conducted more rapidly and a large amount of specimen can be analyzed more efficiently. Therefore, the present invention is very useful in the fields of nucleic acid analyses, clinical tests, and the like utilizing nucleic acid amplification.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-277496 | Oct 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/069371 | 10/24/2008 | WO | 00 | 4/23/2010 |
