JP2006271250A - Method for determination of base sequence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determination of base sequence simple and rapid as compared to conventional method by simply carrying out amplification of a template which is a method utilizing transcribed sequence. <P>SOLUTION: The invention relates to the method for determination of the objective gene sequence by using a product RNA which is obtained in the following processes, a process amplifying the objective gene using strand displacement DNA polymerase and a gene amplifying method, a process carrying out an in vitro transcription reaction using the amplification reaction product DNA as the template and a RNA polymerase and a substrate containing 3'-deoxynucleotide or a labeled 3'-deoxynucleotide. Either one of the internal primers used in above mentioned gene amplification contains a promoter sequence corresponding to the RNA polymerase in the loop part. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for determining a base sequence. More specifically, the present invention relates to a method for determining a nucleotide sequence using a combination of a gene amplification method using a strand displacement type DNA polymerase and an in vitro transcription reaction.

  The development of technology for determining the genetic sequence of organisms has made it possible to analyze the entire genome of an organism together with PCR using thermostable DNA polymerase. Due to this technological development, the entire human genome sequence was decoded in October 2004 [Stein, L.D. Nature, 431: 915 (2004) (Non-patent Document 1)]. However, it has been found that there are many genes whose functions are not known in the actually decoded base sequences, and at present, studies for the purpose of analyzing the functions of the genes are actively conducted. As one of the methods for analyzing the function of this unknown gene, comparative genomics, in which the genome sequence is compared with that of other organisms, has become popular. One example is chimpanzee genome analysis [The international Chimpanzee Chromosome 22 Consortium. Nature, 429 (6990): 382 (2004) (Non-patent Document 2)]. This is an attempt to analogize gene function by comparing the human chimpanzee genome with that of humans. This method makes it possible to analogize the functions of genes related to the characteristic properties of the organisms by comparing genes from various organisms, and is a very effective method. However, it is premised on genome analysis of organisms to be compared, and it is necessary to determine a very large number of base sequences. Therefore, the demand for determining the base sequence will not decrease in the future.

  As a benefit of promoting the Genome Project, automated base sequencing equipment has become popular and easy to use in general laboratories (for example, ABI Prism 3100 Genetic Analyzer manufactured by Applied Biosystems, Inc. Concomitantly, the unit price per data production has decreased, and computers and software that process the base sequence data at high speed have been developed one after another. Is also getting closer.

The base sequence determination method used in the above automatic base sequence determination apparatus is a dideoxy method using a DNA polymerase, but has a drawback that it requires purification of the PCR product prior to the base sequence determination operation. On the other hand, prior to sequencing, a transcriptional sequencing method was developed as a method for directly using the PCR product without purification and obtaining a high-quality sequencing result [Sasaki, N. , et al. Proc. Natl. Acad. Sci. USA, 95 (7): 3455 (1998) (Non-patent Document 3, WO96 / 14434 (Patent Document 1), JP-A-11-75898 (Patent Document 2) WO99 / 02544 (Patent Document 3), JP-A 2003-61684 (Patent Document 4)].
Stein, LD Nature, 431: 915 (2004) The international Chimpanzee Chromosome 22 Consortium. Nature, 429 (6990): 382 (2004) Sasaki, N., et al. Proc. Natl. Acad. Sci. USA, 95 (7): 3455 (1998) WO96 / 14434 JP-A-11-75898 WO99 / 02544 JP 2003-61684 A

  However, in both the dideoxy method and the transcription sequencing method, the template used for base sequence determination must be amplified by PCR or amplified using a host as plasmid DNA. Amplification by PCR requires a thermal cycler and is not suitable for simply amplifying a template.

  Accordingly, the present invention provides a method that uses a transcription sequencing method, which is a new method that can easily amplify a template, and that can determine a base sequence more easily and quickly than conventional methods. The purpose is to do.

  As a DNA amplification method for introducing a promoter sequence necessary for initiating a transcription reaction, the present inventors have used the LAMP method using Bst DNA polymerase (hereinafter abbreviated as “DNAP”) or the like, with the sequence of the amplification target region as a unit. The present invention has been completed by finding out that it can be used as a template for a transcription reaction because it is amplified as DNA fragments of various lengths connected to tandem and a technique for that purpose. The method of the present invention is characterized by rapid base sequence-based characteristics, in combination with the features of the high specificity and high amplification of the LAMP method and the features of the transcription sequencing method using RNA polymerase that do not require purification of the amplification product. Providing genetic analysis methods, it seems to lead to the development of new application fields.

The present invention for solving the above problems is as follows.
[Claim 1] A gene of interest is amplified by a gene amplification method using strand displacement type DNA polymerase, DNA contained in the obtained amplification reaction product is used as a template, RNA polymerase is used, and 3'-deoxynucleotide or labeled In vitro transcription reaction using a substrate containing 3'-deoxynucleotides, and determining the sequence of the target gene using the generated RNA, wherein either one of the internal primers used in the gene amplification method Comprises a promoter sequence corresponding to the RNA polymerase in the loop portion.
[Claim 2] The primer containing the promoter sequence is a forward primer, the number of bases other than the promoter sequence is in the range of 40-50, and the 5 'end of the promoter sequence is 20 from the 5' end of the primer base sequence. The method of claim 1 in the -25th range.
[Claim 3] The primer containing the promoter sequence is a forward primer, the number of bases other than the promoter sequence is 45, and the 5 'end of the promoter sequence is the 21st base from the 5' end of the primer base sequence. The method of claim 1.
[Claim 4] The primer containing the promoter sequence is a backward primer, the number of bases other than the promoter sequence is in the range of 40-50, and the 5 'end of the promoter sequence is from the 5' end of the base sequence of the primer. The method according to any one of claims 1 to 3, which is in the 20th to 25th range.
[5] The primer containing the promoter sequence is a backward primer, the number of bases other than the promoter sequence is 45, and the 5 ′ end of the promoter sequence is the 23rd base from the 5 ′ end of the primer base sequence. The method according to any one of claims 1 to 3.
[6] The method according to any one of [1] to [5], wherein the strand displacement type DNA polymerase is a DNA polymerase derived from Bacillus stearothermophilus.
[7] The method according to any one of [1] to [6], wherein the amplification reaction product is used in an in vitro transcription reaction without being purified.

According to the present invention, a transcription sequence reaction can be performed using a template amplified by the LAMP reaction having a promoter that specifically recognizes RNA polymerase.
Therefore, it is useful when the amount of DNA of the sample to be analyzed is small, and is also useful in a research field where gene base sequencing is performed or a research field where gene analysis is performed.
It is particularly useful when performing genetic analysis based on nucleotide sequences, especially for the purpose of clinical materials and bacterial testing.

  In the method of the present invention, the target gene is amplified by a gene amplification method using a strand displacement type DNA polymerase, an in vitro transcription reaction is performed using the DNA contained in the obtained amplification reaction product as a template, and the generated RNA is obtained. This is a method for determining the sequence of a target gene.

In the conventional transcription sequencing method, the PCR method [Sambrook, J., et al. Molecular Cloning -A Laboratory Manual, 3 ^rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York , 2001]. In contrast, in the present invention, a gene amplification method [LAMP (Loop-mediated Isothermal Amplification) method] using a strand displacement DNA polymerase is used. Unlike PCR, this method uses four unique oligonucleotides as primers on the gene DNA sequence with the target gene sequence in between, and reacts at a constant temperature of 65 ° C, for example. This is a characteristic gene amplification method.

  This gene amplification method (LAMP method) will be described in more detail. PCR requires a denaturation step and a reassociation step for each reaction at the start of the primer reaction for gene amplification. The LAMP method, on the other hand, is designed to form a loop structure that enables the initiation of gene amplification reaction during in vitro reaction with the strand displacement activity (SDA) present in the enzyme used. Four oligonucleotides are used, including a primer (referred to as an internal primer) and a primer (referred to as an external primer) that associates with a specific sequence in the outer gene region. And it is not necessary to raise or lower the temperature to continue this reaction.

  As described above, the LAMP method using primers is 1) high in amplification and 2) highly specific in comparison with PCR [Notomi, T., et al. Nucleic Acids Res. , 28 (12): e63 (2000); JP 2002-330796]. Similarly, the ICAN method [patent registration number 3433929] has been developed as a reaction that can amplify genes at a constant temperature using a DNA polymerase having SDA, but in this case, unlike the LAMP method, Similar to PCR, it uses two oligonucleotides.

  In any case, the gene amplification reaction of the isothermal reaction utilizes the strand displacement type activity inherent in the enzyme, so that no template DNA denaturation work is required, so the gene is amplified as double-stranded DNA. To do.

  By the way, as an applied technology of the LAMP method, it is known to use DNA amplified by the LAMP method as a template for RNA polymerase [Japanese Patent Laid-Open No. 2002-233367]. However, this method uses the LAMP method to prepare a template DNA for RNA expression containing a promoter by carrying out a nucleic acid amplification reaction using a nucleotide chain containing a promoter as an amplification template, and using this promoter as a template It is a method characterized in that RNA is expressed from DNA, and there is no mention about placing a promoter sequence in the primer to enable the transcription reaction to be initiated.

  In the method of the present invention, the purpose is to quickly determine the sequence of a gene. After amplifying the target gene, it is not realistic to add a promoter sequence corresponding to RNA polymerase to the obtained DNA. Absent. Therefore, in the present invention, it was planned to amplify the target gene and simultaneously add a promoter sequence corresponding to RNA polymerase upstream of the target gene without any modification.

  The simplest possible method is to incorporate a sequence complementary to the promoter sequence corresponding to RNA polymerase into the primer used in the amplification method of the target gene. However, unlike the primers used in the PCR method, the primers used in the LAMP method have a very narrow selection range of primer sequences, and even if the primer does not contain extra sequences, its design is different from the primers used in the PCR method. It's not easy. The software for primer design used in the LAMP method has been developed.

  Therefore, as described above, using the LAMP method as the target gene amplification method was expected to have various advantages compared to the case where the target gene was amplified by the PCR method, but it was compatible with RNA polymerase. Whether a target gene can be amplified by the LAMP method using a primer incorporating a complementary sequence of the promoter sequence is unknown, and the design of such a primer has not been published.

  On the other hand, as a result of various studies, the present inventors responded to RNA polymerase upstream by including a promoter sequence corresponding to RNA polymerase in the loop portion of one of the internal primers used in the LAMP method. It was found that a DNA fragment having a target gene sequence including a promoter sequence can be prepared by the LAMP method.

Hereinafter, this point will be described in more detail.
First, the LAMP method was developed to obtain a DNA fragment to which a promoter sequence necessary for transcription initiation was added. For this purpose, it is necessary to quantitatively evaluate the target gene to be amplified using the amplified DNA of the LAMP method, which is said to be at most up to 500 base sequences. For this purpose, several types of plasmids were prepared by cloning fragments of different sizes derived from the λ genome, which is an E. coli phage, into pUC19, an E. coli cloning vector. pUC19 is known as a frequently used gene cloning vector. 1) LAMP primers can be designed using sequences in the vicinity of the cloning site of pUC19, and the LAMP reaction can be evaluated according to the target size. 2) Since the pUC19 vector does not have a promoter sequence, it is possible to evaluate the amplification reaction using the LAMP primer to which the promoter sequence has been added. 3) Since the cloned sequence is a known sequence, the transcription sequence method is used. There are advantages such as being able to evaluate accuracy.

  The structure of the plasmid pULam prepared this time is shown in FIG. The base sequence of all the cloned fragments was determined, and the base sequence in the λ phage genome was confirmed. The lengths of the fragments in the obtained plasmid were 140, 344, 468, 579 and 636 base pairs, and the cloned gene in the figure indicated the number of the λ phage genome sequence.

  A common LAMP primer was designed using the pULam plasmid as a template. Details of the designed primer are shown in FIG. 2 and FIG. FIG. 2 shows the location of sequence-specific hydrogen bonding on the pULam plasmid of the LAMP primer designed this time. The template sequence is shown in SEQ ID NO: 12. The shaded sequences in the internal primer and plasmid sequences in the figure indicate complementary sequences that promote loop formation during the reaction, and each indicates that the corresponding sequence in uppercase and lowercase is a complementary sequence. ing. FIG. 3 shows the entire sequence of the LAMP primer used this time, including an internal primer having a promoter sequence specific to T7 RNA polymerase.

  If a 17-base T7 promoter sequence can be added to a LAMP reaction product, the amplified product should theoretically be usable as a substrate for in vitro transcription reaction. Therefore, in the present invention, a primer is prepared by adding the T7 promoter to the loop portion of the internal primer, and whether or not the LAMP reaction proceeds, and further, the promoter is surely introduced into one end of the amplified target region. We examined whether or not.

  First, two internal primers and two external primers were selected from the primers shown in FIG. 3, four types of primer mixes # 1 to # 4 shown in Table 1 were prepared, and a LAMP reaction was performed. As a result, LAMP reaction was confirmed only with primer mix # 4. Therefore, a forward primer F12a-T7 and a back primer R12a-T7 in which a T7 promoter was introduced into the loop portion of the internal primer used in primer mix # 4 were prepared. And the primer mix of # 5 and # 6 shown in Table 1 was prepared, and LAMP reaction was performed.

  In primer mix # 5 and # 6 using the internal primer with T7 promoter added to the forward and reverse sides, we examined the LAMP reaction by changing various conditions in order to see the effect of the T7 promoter sequence on the LAMP primer. . However, no LAMP reaction was detected under any conditions. In primer mix # 5 and # 6, addition of the T7 promoter sequence was thought to significantly inhibit the LAMP reaction efficiency.

  However, when actually used in a sequencing reaction, the promoter may be added to any one of the target regions, so that primer mixes # 7 and # 8 for adding to the forward and reverse sides were configured, respectively (see Table 1). ). As a result, primer mixes # 7 and # 8 had conditions for causing a LAMP reaction.

  The combinations of LAMP primers shown in Table 1 were shown. As a result of examining the LAMP reaction under various conditions (template amount, primer amount, etc.) for each of them, the LAMP reaction was confirmed in addition to # 4, It was a combination of # 7 and # 8.

  Based on the above results, an in vitro transcription reaction was performed using the LAMP product obtained using primer mix # 7 or # 8 as a template (FIG. 4). The example shown in this figure uses a reaction product that does not require any special purification procedure after the LAMP reaction, and in order to confirm whether the RNA was specifically obtained by in vitro transcription reaction, the transcription reaction product was DNase. And an example of detection by agarose electrophoresis after confirmation by RNase treatment. As a result, in vitro transcripts using LAMP products obtained using the primer mixes of # 7 and # 8 as a template show almost no change in the electrophoretic image in DNase treatment, but when treated with RNase It was decomposed into low molecular weight nucleic acids. Therefore, it was confirmed that a promoter recognized by T7 RNA polymerase was introduced into the LAMP reaction product and functioned sufficiently as a substrate for in vitro transcription reaction.

  Next, using the LAMP product containing the promoter sequence as a template, sequence analysis was performed using a transcription sequencing method. As a transcription sequencing method reagent, CUGA7 Sequencing Kit (manufactured by Nippon Genetech) was used, and the reaction product was analyzed using ABI 310 Genetic Analyzer. As a result, it was shown that sequencing is possible as shown in FIG.

  The following are known strand displacement DNA polymerases. Various mutants of these enzymes can also be used in the present invention as long as they have sequence-dependent complementary strand synthesis activity and strand displacement activity. The term “mutant” as used herein refers to a product obtained by extracting only the structure that provides the catalytic activity required by the enzyme, or a product whose catalytic activity, stability, or heat resistance has been modified by amino acid mutation or the like. Bst DNA polymerase, Bst DNA polymerase large fragment (enzyme used in LAMP method, lacking 5'-3 'exonuclease activity), Bca (exo-) DNA polymerase, E. coli DNA polymerase I Klenow Fragment, Vent DNA polymerase, Vent (Exo-) DNA polymerase (Vent DNA polymerase minus 5'-3 'exonuclease activity), DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, (5 from DeepVent DNA polymerase Excluding '-3' exonuclease activity), Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase (Takara Shuzo), KOD DNA polymerase (Toyobo).

  Among these enzymes, Bst DNA polymerase / large fragment and Bca (exo-) DNA polymerase are particularly desirable enzymes because they have a certain degree of heat resistance and high catalytic activity. The reaction of the present invention can be carried out isothermally in a desirable embodiment, but a temperature condition suitable for enzyme stability is not always available for adjusting the melting temperature (Tm) and the like. Therefore, it is one of desirable conditions that the enzyme is thermostable. Although isothermal reaction is possible, heat denaturation may be performed to provide the initial template nucleic acid. In this respect, the use of thermostable enzymes broadens the choice of assay protocol. .

  Vent (Exo-) DNA polymerase is an enzyme with high thermostability as well as strand displacement activity. By the way, it is known that the complementary strand synthesis reaction involving strand displacement by DNA polymerase is promoted by the addition of a single strand binding protein (Paul M. Lizardi et al, Nature Genetics 19, 225). -232, July, 1998). By applying this action to the present invention and adding a single-stranded binding protein, an effect of promoting complementary strand synthesis can be expected. For example, T4 gene 32 is effective as a single-stranded binding protein for Vent (Exo-) DNA polymerase.

  For DNA polymerases that do not have 3'-5 'exonuclease activity, it is known that complementary strand synthesis does not stop at the 5' end of the template, but proceeds to a state where one base protrudes. Yes. In the present invention, such a phenomenon is undesirable because the sequence at the 3 ′ end when complementary strand synthesis reaches the end leads to the start of the next complementary strand synthesis. However, the addition of a base to the 3 ′ end by DNA polymerase is A with a high probability. Therefore, the sequence may be selected so that synthesis from the 3 ′ end starts with A so that no problem is caused even if dATP is mistakenly added with one base. In addition, even if the 3 ′ end protrudes during the synthesis of the complementary strand, the 3 ′ → 5 ′ exonuclease activity can be used by digesting this to make a blunt end. For example, since natural Vent DNA polymerase has this activity, this problem can be avoided by mixing with Vent (Exo-) DNA polymerase.

A series of reactions consist of a buffer that gives a suitable pH for the enzyme reaction, salts necessary for maintaining the catalytic activity of the enzyme and annealing, an enzyme protecting agent, and a melting temperature (Tm) adjusting agent as necessary. It is appropriate to carry out in the coexistence of the above. As the buffering agent, a neutral to weakly alkaline buffering agent such as Tris-HCl is appropriately used. The pH is appropriately adjusted according to the DNA polymerase used. As salts, KCl, NaCl, (NH ₄ ) ₂ SO _4, or the like is appropriately added for maintaining the activity of the enzyme and adjusting the melting temperature (Tm) of the nucleic acid. As the enzyme protective agent, for example, bovine serum albumin or saccharide is used.

  Furthermore, betaine (N, N, N, -trimethylglycine), proline, dimethyl sulfoxide (hereinafter abbreviated as DMSO), or formamide are used as regulators for the melting temperature (Tm) of the primer and template DNA during the DNA polymerase reaction. Is generally used. By using a melting temperature (Tm) adjusting agent, the annealing of the oligonucleotide can be adjusted under limited temperature conditions. Furthermore, betaine and tetraalkylammonium salts are also effective in improving the strand displacement efficiency by their isostabilize action. Betaine can be expected to promote the nucleic acid amplification reaction of the present invention by adding 0.2 to 3.0 M, preferably about 0.5 to 1.5 M, in the reaction solution. These melting temperature regulators act in the direction of lowering the melting temperature, so appropriate conditions for giving appropriate stringency and reactivity should be set in consideration of other reaction conditions such as salt concentration and reaction temperature. Can do.

  By using the Tm adjusting agent, it is possible to easily set a temperature condition suitable for the enzyme reaction. Tm varies depending on the relationship between the primer and the target base sequence. Therefore, it is desirable to adjust the use amount of the Tm adjusting agent so that the conditions under which the enzyme activity can be maintained and the conditions for incubation satisfying the conditions of the present invention match. Based on the disclosure of the present invention, it is obvious to those skilled in the art to set an appropriate amount of Tm regulator to be used according to the base sequence of the primer. For example, Tm can be calculated based on the length of the base sequence to be annealed, its GC content, salt concentration, and Tm regulator concentration.

  It is speculated that annealing of a primer to a double-stranded nucleic acid under such conditions is probably unstable. However, by incubating with a strand displacement type polymerase, a complementary strand is synthesized using a primer that has been annealed in an unstable manner as a replication origin. If the complementary strand is synthesized, primer annealing will be gradually stabilized.

Relationship between reaction time and melting temperature
In the reaction in the LAMP method, the following components are added to a single-stranded nucleic acid as a template, and the base sequence constituting each primer can form a stable base pair bond with a complementary base sequence. Can proceed and proceed by incubating at a temperature that can maintain enzyme activity. The incubation temperature is 50 to 75 ° C, preferably 55 to 70 ° C, and the incubation time is 1 minute to 10 hours, preferably 5 minutes to 4 hours.

  The promoter sequence corresponding to the RNA polymerase included in the internal primer can be appropriately determined according to the type of RNA polymerase. The RNA polymerase used in the transcription sequencing method can be a wild type and a mutant RNA polymerase, and are described in, for example, JP-A Nos. 11-75867 and 2003-61683. Examples of the promoter sequence include T7 polymerase promoter, hsp16-2 promoter, sv40 promoter, and CMV promoter.

  In the present invention, the internal primer includes a promoter sequence corresponding to the RNA polymerase in the loop portion. Specifically, the primer containing the promoter sequence is a forward primer, the number of bases other than the promoter sequence is in the range of 40-50, and the 5 ′ end of the promoter sequence is 20 from the 5 ′ end of the primer base sequence. It can be in the ˜25th range. In particular, the primer containing the promoter sequence is a forward primer, the number of bases other than the promoter sequence is 45, and the 5 ′ end of the promoter sequence is the 21st base from the 5 ′ end of the primer base sequence. it can.

  The internal primer containing a promoter sequence corresponding to the RNA polymerase in the loop part is a primer containing the promoter sequence is a backward primer, the number of bases other than the promoter sequence is in the range of 40-50, The 5 'end can be in the 20th to 25th range from the 5' end of the base sequence of the primer. In particular, the primer containing the promoter sequence is a backward primer, the number of bases other than the promoter sequence is 45, and the 5 ′ end of the promoter sequence is the 23rd base from the 5 ′ end of the primer base sequence. Can do.

  The sequence other than the promoter sequence in the internal primer (original primer sequence) and the sequence of the external primer can be appropriately determined in consideration of the sequence of the template.

  In the method of the present invention, a transcription sequencing method (transcription reaction, electrophoresis, and reading of a DNA region), an RNA polymerase used in the transcription sequencing method, a 3′-deoxynucleotide that is a substrate or terminator of RNA polymerase, or a labeled 3 ′ Known deoxynucleotides can be used as they are. For example, for transcription sequencing methods, WO96 / 14434, JP-A-11-75898, WO99 / 02544, JP-A-2003-61684, and RNA polymerase As for the terminator of 11-75867, JP-A-2003-61683, and the terminator, those described in JP-A-11-80189 can be used.

The DNA base sequence determination method of the present invention comprises (1) a step of obtaining a nucleic acid transcription reaction product using RNA polymerase, a template DNA having a promoter sequence for the RNA polymerase, and a substrate for the RNA polymerase, (2) Separating the obtained nucleic acid transcription reaction product, and (3) reading a nucleic acid sequence from the obtained separated fraction.
Using a DNA fragment containing a promoter sequence for RNA polymerase as a template, a method for enzymatically synthesizing a nucleic acid transcription product using RNA polymerase, a method for separating a nucleic acid transcription product, and a method for separating nucleic acid from a separated fraction The method for reading the sequence is in principle any known method except for a method for preparing a template containing a DNA fragment containing a promoter sequence for RNA polymerase. Therefore, basically, any known method, conditions, apparatus, and the like can be used as appropriate regarding these points.

(1) Step of obtaining nucleic acid transcription reaction product The DNA fragment used as a template is not particularly limited except that it contains a promoter sequence for RNA polymerase. For example, a DNA fragment containing a promoter sequence can be a DNA product amplified by the polymerase chain reaction. Furthermore, the nucleic acid transcription reaction in the method of the present invention can be carried out without removing the primer and / or 2 ′ deoxyribonucleoside 5 ′ triphosphate and / or its derivative used in the polymerase chain reaction from the amplified DNA product. It can be carried out. For the reaction for DNA amplification, a method widely used as a PCR method can be used as it is. In addition, the DNA fragment containing the promoter sequence may be a DNA fragment cloned using a suitable host after ligating the promoter sequence and the DNA fragment to be amplified. That is, in the present invention, there are no particular limitations on the DNA sequence, primers, amplification conditions, and the like that are to be amplified.

  For example, as a polymerase chain reaction reaction system for amplification of a DNA fragment containing a promoter sequence, 10-50 ng genomic DNA or 1 pg cloned DNA, 10 μM each primer, 200 μM each 2 ′ deoxyribonucleoside 5 ′ trifo For example, Taq polymerase can be used as a DNA polymerase in a 20 μl volume containing sulfate (dATP, dGTP, dCTP, dTTP).

  However, either one of the primers for the polymerase chain reaction or the amplified insert DNA needs to contain a promoter sequence for RNA polymerase described later. The direct transcription sequence method can be obtained by using a primer having a phage promoter sequence in one of two kinds of primers in the PCR method or by having a phage promoter sequence in the amplified inserted DNA. The PCR product can be subjected to in vitro transcription using RNA polymerase working by its promoter.

  The promoter sequence for RNA polymerase can be appropriately selected depending on the type of RNA polymerase used. However, two RNA-derived promoters, for example, two promoters specifically recognized by T7 and T3 phages, are introduced into each strand of the template so that only one RNA polymerase can be selected during the transcription sequence reaction. It is possible to analyze the sequence data of both ends of this target DNA.

  In the method of the present invention, a nucleic acid transcript such as an RNA transcript is synthesized from a DNA fragment containing a promoter sequence by the LAMP method. Since the DNA fragment contains a promoter sequence for RNA polymerase, this promoter sequence activates the mutant RNA polymerase to synthesize a nucleic acid transcript such as an RNA transcript.

  For the synthesis of a nucleic acid transcript such as an RNA transcript, a substrate containing at least 3′-deoxynucleotide or fluorescently labeled 3′-deoxynucleotide (3′dNTP derivative) is used as a substrate for a mutant RNA polymerase described later. More specifically, the substrate uses ribonucleoside 5 'triphosphates (NTPs) composed of ATP, GTP, CTP and UTP or derivatives thereof, and one or more 3' dNTP derivatives. In the present specification, the 3'dNTP derivative is used as a general term for 3'dATP, 3'dGTP, 3'dCTP, 3'dUTP and derivatives thereof. As ribonucleoside 5 'triphosphates (NTPs), at least four kinds of compounds having different bases are required for the synthesis of transcripts, including the case where some of them are derivatives such as ATP. However, two or more compounds containing the same base can be used.

  By incorporating a 3'dNTP derivative into the 3 'end of RNA or nucleic acid that is a transcription product, the 3' hydroxy group is lost and the synthesis of RNA or nucleic acid is inhibited. As a result, RNAs or nucleic acid fragments of various lengths having a 3 'end that is a 3'dNTP derivative are obtained. Such ribonucleoside analogs are obtained for each of the four types of 3'dNTP derivatives having different bases. By preparing four ribonucleoside analogs, it can be used to determine RNA or nucleic acid sequences [Vladimir D. Axelred er al. (1985) Biochemistry Vol. 24, 5716-5723].

  One or more 3'dNTP derivatives can be used for one nucleic acid transcription reaction. When one nucleic acid transcription reaction is carried out using only one kind of 3′dNTP derivative, four transcription products with different bases of the 3′dNTP derivative at the 3 ′ end are obtained by performing the nucleic acid transcription reaction four times. . In one nucleic acid transcription reaction, a transcription product is obtained which is a mixture of various RNAs or nucleic acid fragments having the same 3'dNTP derivative at the 3 'end and different molecular weights. The obtained four transcription products can be independently subjected to separation and sequence reading described later. It is also possible to mix two or more of the four transcription products and to use this mixture for separation and sequence reading.

  When two or more kinds of 3 ′ dNTP derivatives are used simultaneously in one nucleic acid transcription reaction, two or more transcription products having different bases of the 3 ′ dNTP derivative at the 3 ′ end are contained in one reaction product. It will be. This can be used for separation and array reading described below. It is preferable to use two or more 3 ′ dNTP derivatives simultaneously in the nucleic acid transcription reaction because the number of nucleic acid transcription reaction operations can be reduced.

  In the present invention, it is particularly preferable that transcription of nucleic acid such as RNA is terminated by four kinds of 3'dNTP derivatives having different bases, which are separated and the sequences of the four kinds of bases are read at the same time (simultaneously).

(2) Step of separating nucleic acid transcription reaction product In the method of the present invention, RNA or nucleic acid transcription product is separated. This separation can be appropriately performed by a method capable of separating a plurality of product molecules having different molecular weights contained in the transcription product according to the molecular weight. Examples of such a separation method include electrophoresis. In addition, HPLC or the like can also be used.

  There are no particular limitations on the conditions of electrophoresis and the like, and the electrophoresis can be performed by a conventional method. The sequence of RNA or nucleic acid can be read from a band (RNA or nucleic acid ladder) obtained by subjecting the transcription product to electrophoresis.

(3) Step of reading nucleic acid sequence RNA or nucleic acid ladder can be read by labeling a 3′dNTP derivative. RNA or nucleic acid ladder can also be read by labeling ribonucleoside 5 ′ triphosphates (NTPs). Examples of the label include radioactive or stable isotopes or fluorescent labels.

  Specifically, for example, a labeled 3′dNTP derivative, more specifically, labeled 3′dATP, 3′dGTP, 3′dCTP, and 3′dUTP are used to subject the transcription product to electrophoresis. The sequence of the transcription product can be read by detecting the radioactive or stable isotope or fluorescence of the band obtained as described above. By labeling the 3'dNTP derivative in this manner, there is no variation in the radioactive intensity or fluorescence intensity between any bands, and the measurement becomes easy. Furthermore, detection of a ladder that generates radioactive or stable isotopes or fluorescence can be performed appropriately using, for example, an apparatus used for determining the base sequence of DNA (such an apparatus is generally called a DNA sequencer).

  Reading the sequence of transcripts by detecting radioactive or stable isotopes or fluorescence of bands obtained by electrophoresis using radioactive or stable isotopes or fluorescently labeled ATP, GTP, CTP and UTP Can do.

  Furthermore, 3′dATP, 3′dGTP, 3′dCTP and 3′dUTP labeled with different fluorescence are used, and the ends are 3′dATP, 3′dGTP, 3′dCTP or 3′dUTP, and different labels are made. The sequence of RNA or nucleic acid can also be read by detecting four types of fluorescence of bands obtained by subjecting a mixture of various transcription product fragments to electrophoresis.

  In this method, four types of 3'dNTPs are labeled with different fluorescences. In this way, by subjecting a mixture of four types of transcription products having different 3 ′ ends to electrophoresis, bands emitting fluorescence corresponding to four different 3 ′ dNTPs at the 3 ′ end can be obtained. While distinguishing this difference in fluorescence, it is possible to read the sequence of four types of RNA or nucleic acid at a time. As the fluorescently labeled 3'dNTP, for example, 3 'deoxyribonucleotide derivatives described in JP-A-11-80189 and WO99 / 02544 are preferably used.

[Example 1] Construction of pULAN
In order to evaluate whether a template amplified by the LAMP method can be used as a template for cycle sequencing or transcription sequencing, it is said that the size of the sequence to be amplified is about 500 base pairs at most, and quantitative evaluation is required. It is. Therefore, fragments of different lengths derived from E. coli phage λ phage DNA were cloned into E. coli cloning vector pUC19 as known sequence DNA, and several types of plasmids were prepared as follows. Unless otherwise specified, the experimental method and the buffer used were described in [Sambrook, J., et al. Molecular Cloning -A Laboratory Manual, 3 ^rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York , 2001] or made.

  A DNA fragment of a known sequence to be cloned is obtained by cleaving λ phage genomic DNA with restriction enzyme PvuII (Nippon Gene), and performing electrophoresis with 1% agarose L (Nippon Gene) to obtain a DNA fragment of 100 to 1000 base pairs. The agarose fragment containing was excised, purified using Gene Pure (manufactured by Nippon Gene), and finally dissolved in 20 μl of TE buffer. The DNA fragment prepared in this way is cleaved with the restriction enzyme HincII (Nippon Gene), mixed with dephosphorylated pUC19 DNA, and attached to the kit using a ligation kit manufactured by Takara Bio. Ligation reaction was performed according to the protocol. The reaction volume is 8μl, performed at 16 ° C for 30 minutes, 1μl of which is introduced into E. coli DH5a by electroporation, applied to an agar plate medium containing IPTG, X-gal and ampicillin, and kept at 37 ° C overnight. Many transformants of white colonies were obtained. Take a small amount of this transformant with a toothpick and perform PCR reaction using FPuc1 (5'-CTGCAAGGCGATTAAGTTGG-3 ') (SEQ ID NO: 13) and RPuc1 (5'-GCTATGACCATGATTACGCC-3') (SEQ ID NO: 14) as primers. (This method is called colony PCR because it uses the cells collected from the colony directly). This PCR product was subjected to 1% agarose electrophoresis, and a large number of transformants having a size larger than that of the PCR product obtained from the blue colony could be obtained. These are all expected clones containing DNA derived from λ phage DNA, but when plasmid DNA was extracted from transformed colonies and the base sequence of the cloned DNA fragment was determined, all were λ phage. It was confirmed that the sequence was derived from DNA. The resulting plasmid was named pULam and, in order of the size of its cloned DNA fragment, was pULam4, 3, 2, 12 and 14, and the insert sizes were 140, 344, 468, 579, respectively. And 636 base pairs (FIG. 1).

[Example 2] Design of primers used in the LAMP method using pLam DNA as a template
The pULam DNAs differ from each other only in the insert size, and the other sequences are common. A LAMP primer sandwiching the insert DNA was designed. The purpose of this design is to finally design a LAMP primer into which a promoter sequence has been introduced. Here, the primer for introducing the promoter sequence was in principle the loop portion of the internal primer.

  FIG. 2 shows the location of sequence-specific hydrogen bonding on the pULam plasmid DNA of the LAMP primer designed this time. The shaded sequences in the internal primer and plasmid sequences in the figure indicate complementary sequences that promote loop formation during the reaction, and each indicates that the corresponding sequence in uppercase and lowercase is a complementary sequence. Have (for example, R1a and r1a). FIG. 3 shows the entire sequence of the LAMP primer used this time, including an internal primer specific to T7 RNA polymerase.

  The actual design is to first select the combination that gave the LAMP reaction product using the LAMP primer that does not contain the promoter sequence, and then specifically recognize the T7 RNA polymerase consisting of 17 base pairs in the loop part of the internal primer. A promoter sequence was inserted.

Specifically, it is as follows. Using pULam4 DNA as a template, pULam4DNA 1ng, 20 mM Tris-HCl, pH 8.0, 10 mM KCl, 10 mM (NH ₄ ) SO ₄ , 2 mM MgSO ₄ , 0.1% Triton-X, 1.6 μM Forward inner primer In the reaction system (25μL) of 1.6μM Backward inner primer (The first letter of the inner primer is expressed as R), 0.4μM Forward outer primer (F3), 0.4μM Backward outer primer (R3) Before the addition of DNA polymerase, the mixture was treated at 95 ° C. for 5 minutes, rapidly cooled to 4 ° C., 8 units of Bst DNA polymerase large fragment (NEB) was added, and immediately reacted at 60 ° C. for 1 hour. After completion of the reaction, 1% agarose gel electrophoresis was performed. After the electrophoresis, staining was performed with ethidium bromide (final concentration 100 μg / ml), and it was confirmed by the swimming image whether LAMP was proceeding well. As a result, amplification was observed only in LAMP primer mix # 4. Therefore, a T7 promoter sequence consisting of 17 bases was introduced into the loop portions of F12a and R12a of the internal primer used in this combination, and F12a-T7 and R12a-T7 added respectively were designed. After this study, it was found that amplification occurs when LAMP primer mixes # 7 and # 8 containing an internal primer containing a T7 RNA polymerase sequence are used. In this case, it was found that when a primer having a promoter sequence was used at the above-mentioned reaction concentration, amplification was not observed and amplification was performed at a final concentration of 3.6 μM. Table 1 shows all LAMP primer combinations examined this time. In addition, this table shows the combination and the results of the LAMP reaction when the combination was used. As a result, LAMP reaction was observed at # 4, # 7 and # 8, and it was found that LAMP reaction with a promoter sequence inserted could be performed.

  PULam4, 3, 2, 12 and 14 DNAs were 140, 344, 468, 579 and 636 base pairs, respectively, and it was confirmed that LAMP reaction can be performed.

[Example 3] In vitro transcription reaction using pULam
Using pULam4 DNA as a template, LAMP reaction was performed with LAMP primer mix # 7 and # 8 in a reaction volume of 20 μl. The final concentration of the internal primer containing the promoter sequence used in this reaction is 3.6 μM. The reaction was performed at 60 ° C. for 1 hour, and it was confirmed whether 1 μl from the reaction solution was amplified by 1% agarose electrophoresis. After confirming the amplification, 4 μl was taken from the same reaction solution, and reacted for 1 hour at 37 ° C. with 20 units of T7 RNA polymerase (Invitrogen), reaction volume of 40 μl. After the reaction is complete, divide the solution into 3 new plastic tubes of 10 µl each, add 1 µl of DNase or RNase A, and incubate at 37 ° C for 30 minutes. After completion of the reaction, all the reaction products were subjected to 1% agarose electrophoresis, and it was confirmed whether in vitro transcription was possible using the LAMP method amplification product as a template. As a result, when treated with RNase A, a nucleic acid that significantly decreases in molecular weight is detected, and thus it can be seen that this is RNA. However, this molecule was not observed when T7RNAP was not added, and it was confirmed that the LAMP amplification product using the LAMP primer into which the promoter sequence was introduced was indeed a substrate for in vitro transcription.

[Example 4] Transcription sequence reaction with LAMP amplification product using pULam as template
First, it was examined whether the nucleotide sequence could be determined using the LAMP amplification product into which the promoter sequence obtained by the LAMP reaction was introduced as a template for the cycle sequencing method. The LAMP reaction is performed under the same conditions as described in Example 3, and half of the reaction solution (10 μl) is purified using Monoface DNA Purification Kit I (manufactured by GL Sciences) and finally attached to the kit. The purified DNA solution was eluted with 10 μl of the eluted solution. Of this eluate, 1 μl was used, and a cycle sequence reaction was performed using a cycle sequence kit (BigDye Kit). After the reaction, the reaction product was purified using Qiagen Kit, and the base sequence was determined using ABI 3100 genetic analyzer. As a result, it was confirmed that it was a partial sequence of the λ phage genome, which is an insert DNA of pULam4. Therefore, it was found that the template amplified by the LAMP method can be used as a cycle sequence template.

  Moreover, transcription sequence reaction was performed using 2 μl of the remaining reaction solution. The reaction was carried out according to the manual protocol using CUGA7 Sequencing Kit for ABI PRISM 310 Genetic Analyzer (Nippon Gene), which is a reagent kit for transcription sequencing. This kit contains T7 RNA polymerase that recognizes the T7 promoter. After the transcription sequence reaction, the sample was purified by the ethanol precipitation method recommended in the manual and analyzed using the ABI 310 Genetic Analyzer. The result is shown in FIG. From this result, it was shown that a transcription sequence using a template amplified by the LAMP method is possible.

  The present invention is useful in a research field in which a base sequence of a gene is determined or a research field in which a gene analysis is performed. It is particularly useful when performing genetic analysis based on nucleotide sequences, especially for the purpose of clinical materials and bacterial testing.

The structure of the prepared plasmid pULam is shown. The location of the designed LAMP primer on the pULam plasmid for sequence-specific hydrogen bonding is shown. All sequences of the LAMP primer used this time, including an internal primer with a promoter sequence specific for T7 RNA polymerase, are shown. Results of in vitro transcription reaction using the LAMP product obtained using primer mix # 7 or # 8 as a template (electrophoresis photograph). The analysis result of the transcription sequence reaction product in Example 4 using ABI 310 Genetic Analyzer.

Claims (7)

Amplify the target gene by gene amplification method using strand displacement DNA polymerase,
Using the DNA contained in the obtained amplification reaction product as a template, using RNA polymerase, and performing an in vitro transcription reaction using a substrate containing 3′-deoxynucleotide or labeled 3′-deoxynucleotide,
A method of determining the sequence of a target gene using generated RNA,
One of the internal primers used in the gene amplification method comprises a promoter sequence corresponding to the RNA polymerase in a loop portion. The primer including the promoter sequence is a forward primer, and the number of bases other than the promoter sequence is in the range of 40 to 50, and the 5 ′ end of the promoter sequence is in the 20th to 25th range from the 5 ′ end of the primer base sequence. The method according to claim 1. The primer containing a promoter sequence is a forward primer, the number of bases other than the promoter sequence is 45, and the 5 ′ end of the promoter sequence is the 21st base from the 5 ′ end of the base sequence of the primer. The method described. The primer containing the promoter sequence is a backward primer, and the number of bases other than the promoter sequence is in the range of 40-50, and the 5 ′ end of the promoter sequence is 20th to 25th from the 5 ′ end of the primer base sequence. 4. A method according to any one of claims 1 to 3 in the range. The primer containing a promoter sequence is a backward primer, the number of bases other than the promoter sequence is 45, and the 5 'end of the promoter sequence is the 23rd base from the 5' end of the primer base sequence. The method of any one of -3. The method according to any one of claims 1 to 5, wherein the strand displacement type DNA polymerase is a DNA polymerase derived from Bacillus stearothermophilus. The method according to any one of claims 1 to 6, wherein the amplification reaction product is used for in vitro transcription reaction without purification.