JP4304350B2 - Polynucleotide synthesis method - Google Patents

Description

【図面の簡単な説明】
【図１】実施例に用いたMarathon cDNA amplification kitの反応原理を示す図である。図中、１はアダプタープライマーのアニール位置、２はnested PCRのためのアダプタープライマーがアニールする位置を示した。ds cDNAは２本鎖cDNAを示し、またNNT30は、cDNAの合成に用いられたプライマーを示す。
【図２】マウスMsh4遺伝子に5'RACE法を応用して得られたPCR産物の電気泳動結果を示す写真である。1stは5'側のプライマーを用いた場合の結果を、2ndaryは3'側のプライマーを用いた場合の結果を示す。
【図３】マウスβアクチン遺伝子に5'RACE法を応用して得られたPCR産物の電気泳動結果を示す写真である。
【図４】アダプタープライマーを用いないDNA合成のメカニズムを示す図。
【図５】アダプタープライマーを用いないPCR産物の電気泳動結果を示す写真である。
【図６】本発明によって増幅されたDNAの末端部分の塩基配列を決定した結果を示す図である。図中、プライマーの塩基配列を四角で囲んで示した。また末端部分に付加されたプライマーに由来しに相補的な塩基配列にアンダーラインを付けた。各塩基配列はスプライシングバリアントごとに記載され、上が5'末端の、下が3'末端の塩基配列を示す。各塩基配列の左側にスプライシングバリアントの名前と、増幅産物の長さを示した。
【図７】本発明によって増幅されたDNAの末端部分の塩基配列を決定した結果を示す図である（図６の続き）。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a polynucleotide amplification method.
[0002]
[Prior art]
In the isolation of a polynucleotide, the method for amplifying the polynucleotide is one of the important steps. The most common method for amplifying a polynucleotide is a PCR method (Polymerase Chain Reaction). The DNA synthesis reaction by the PCR method is composed of repetition of complementary strand synthesis reaction in the 3 ′ direction from the primer. In the PCR method, newly synthesized DNA is used as a template after denaturation, so that the reaction product increases exponentially. The primer used in the PCR method has a complementary base sequence in the 5 ′ region of the target base sequence. Therefore, at least the base sequence of the portion where the primer anneals must be known in advance. In other words, DNA whose base sequence is unknown cannot be selectively amplified by PCR.
[0003]
However, selective amplification is necessary even for DNA whose base sequence is unknown. For example, acquisition of the full length of a gene based on a partial base sequence is often attempted. Specifically, the 5 ′ region of mRNA is difficult to synthesize as cDNA. For this reason, cDNAs with incomplete 5 ′ base sequences are often cloned. In order to clarify the full-length nucleotide sequence of an incomplete cDNA lacking the 5'-side nucleotide sequence, it is necessary to selectively amplify DNA containing the 5'-side nucleotide sequence consisting of an unknown nucleotide sequence. .
[0004]
Further, in the differential display method, which is a method for obtaining a gene having a specific function, as a result, a fragment base sequence of the gene is obtained. If the obtained base sequence does not match a known gene, the full-length base sequence of the gene must be clarified. At this time, acquisition of the full length of the gene based on the fragment base sequence is attempted.
[0005]
There is known a method for obtaining the full length of a gene based on this partial base sequence when only a part of the base sequence constituting the gene is clear. A typical method is the RACE (Rapid Amplification of cDNA Ends) method. The RACE method is a method of amplifying a region containing an unknown base sequence using PCR with a gene whose terminal base sequence is unknown as a template. Primers for PCR are designed using a portion whose base sequence is clear. If the base sequence of the obtained PCR product is determined, the unknown base sequence becomes clear. There are several known variations of the RACE method, such as a method for adding a region for the primer to anneal.
[0006]
For example, a kit for the RACE method commercially supplied by Clonetech as the SMART (Switching Mechanism At 5 'end of RNA Transcript) method is based on the following principle. The synthesis of the contained cDNA has been realized (Nucleic Acids Res. 27/6, 1558-1560, 1999). The SMART method utilizes the special enzyme activity of reverse transcriptase used for the synthesis of the first strand of cDNA. In the SMART method, a mutant of reverse transcriptase called MMLVRT (Moloney murine leukemia virus reverse transcriptase) is used.
[0007]
This reverse transcriptase continuously adds the same base to the 3 ′ end of the synthesized cDNA after the complementary strand synthesis reaches the 5 ′ end of RNA as a template. The base added is often c. Therefore, an oligonucleotide having a base sequence having g continuous as a base sequence complementary to the c stretch at the 3 ′ end serves as a primer that anneals to the 3 ′ end of cDNA synthesized by MMLVRT.
[0008]
The SMART method is a primer for synthesis of cDNA second strand (SMART oligo), an oligonucleotide that has a g chain at the 3 'end and a special base sequence called SMART sequence at the 5' end. Is used. When the g chain arranged at the 3 ′ end of “SMART oligo” anneals to the c chain of cDNA, reverse transcriptase also synthesizes a complementary strand for the 5 ′ SMART sequence portion of “SMART oligo”. Thus, the base sequence of “SMART oligo” is added to the end of the cDNA as a complete double strand.
[0009]
On the other hand, “SMART oligo” itself functions as a primer for complementary strand synthesis using the first strand of cDNA as a template. As a result, the second strand of cDNA is also synthesized, and a double-stranded cDNA can be obtained. This cDNA can be amplified by PCR using a SMART sequence arranged on the 5 ′ side of “SMART oligo” and a gene-specific primer used for cDNA synthesis. When an oligonucleotide having a length that allows two primers to be set is used as the “SMART oligo”, nested PCR can be applied.
[0010]
There are several variations of the RACE method besides the SMART method. For example, Marathon-ready cDNA, a RACE method kit commercially supplied by Clontech, realizes the addition of a known base sequence to the end based on a principle different from the SMART method. Marathon-ready cDNA utilizes a double-stranded oligonucleotide called “Marathon adaptor”. The “Marathon adaptor” is composed of a short oligonucleotide that does not become a blocked primer at the 3 ′ end, and a long oligonucleotide that has a base sequence complementary to this oligonucleotide at the 3 ′ end.
[0011]
The 5 ′ side of the short oligonucleotide in the “Marathon adaptor” is blunt ended. By ligating the blunt end of “Marathon adaptor” to the end of cDNA, DNA whose base sequence is known is added to the unknown base sequence. If PCR is performed using the ligation product as a template and the following primer set, DNA containing an unknown base sequence can be amplified.
-Gene-specific primer that anneals to a known base sequence
-Primer having a base sequence complementary to the 5 'base sequence of a long oligonucleotide composing "Marathon adaptor" (region not overlapping with short oligonucleotide)
[0012]
In the SMART method or Marathon-ready cDNA principle, the same base sequence may be added to both ends of the cDNA. In these methods, by using suppression PCR (Diatchenko L, et al., Proc Natl Acad Sci USA 1996 Jun, 11; 93 (12): 6025-30), the same nucleotide sequence is present at both ends. It suppresses the synthesis of added cDNA.
[0013]
Suppressive PCR uses a phenomenon in which cDNAs having the same base sequence added at both ends are difficult to synthesize in the PCR method. A cDNA to which the same base sequence is added is composed of base sequences complementary to each other at the 5 ′ end and 3 ′ end of each strand (inverted repeat). As a result, in the step of denatured into a single strand to become a template, complementary base sequences at both ends hybridize with each other to take a frying pan-like structure. Since hybridization within the same strand occurs preferentially over hybridization with a primer, a template having such a structure cannot be a template for PCR. This is the principle of suppression PCR.
[0014]
Other variations of the RACE method are known. For example, even for DNA having an unknown base sequence at the terminal portion, PCR can be designed using a portion whose base sequence is known by cyclizing both ends. This method has no specificity for the 5 ′ end of the mRNA. Therefore, it is not suitable particularly when trying to obtain the full length of a gene whose 5 ′ side is unknown.
A method of linking an oligonucleotide having a known base sequence to the 3 ′ end of the first strand of cDNA is also known. This method requires a step of purifying the first strand after synthesis.
Therefore, it can be said that the SMART method and Marathon-ready cDNA are advantageous methods that can be expected to have specificity for the 5 ′ end of mRNA. In particular, the SMART method uses fewer enzymes and the reaction process is simpler than other methods.
[0015]
However, although it can be expected to have characteristics for the 5 ′ end of mRNA, it is true that it is still difficult to obtain the full length of a gene with a low transcription amount. Therefore, it would be useful if a new RACE method capable of obtaining the full length of a small amount of mRNA was provided.
[0016]
[Problems to be solved by the invention]
An object of the present invention is to provide a new principle for synthesizing a polynucleotide whose base sequence in the region including the 5 ′ end is unknown.
[0017]
[Means for Solving the Problems]
In the RACE method, the target gene is finally synthesized using a primer-dependent complementary strand synthesis reaction such as PCR. In general, in the primer-dependent complementary strand synthesis reaction, the base sequence of the primer must be designed based on the base sequence of the polynucleotide to be synthesized. On the other hand, the purpose of the RACE method is to synthesize a region whose base sequence is unknown. Therefore, what kind of base sequence is selected as the base sequence to be annealed by the primer, and how the base sequence is introduced is a major factor in determining the performance of the RACE method. However, in the known RACE method, what kind of base sequence can be introduced to obtain a desired result has not been sufficiently studied. Moreover, it can be said that there is still room for improvement in the method of introducing a base sequence.
[0018]
In contrast, in the synthesis of a complementary strand of a template polynucleotide containing an unknown base sequence on the 5 ′ side, the present inventors added a specific base sequence to the 3 ′ end of the synthesized complementary strand, thereby adding RACE. We found that the performance of the law can be expected to improve. Furthermore, the present invention was completed by clarifying reaction conditions suitable for this purpose. That is, the present invention relates to the following method.
[1] A method for synthesizing a polynucleotide whose base sequence in the region containing the 5 ′ end is unknown, comprising the following steps.
a) Step of synthesizing a complementary strand using an oligonucleotide that anneals to an arbitrary region of the polynucleotide as a primer
b) adding a base sequence complementary to the oligonucleotide of step a) to the 3 ′ end of the complementary strand synthesized in step a); and
c) using the complementary strand obtained in step b) as a template, synthesizing a polynucleotide whose base sequence of the region including the 5 ′ end is unknown using the oligonucleotide of step a) as a primer,
[2] The method according to [1], wherein step b) comprises a step of incubating the following elements under conditions allowing a complementary strand synthesis reaction.
(1) Enzyme that catalyzes template-dependent complementary strand synthesis reaction
(2) the polynucleotide synthesized in step a), and
(3) Nucleotide substrate
[3] The method according to [1], wherein the polynucleotide having an unknown base sequence in the region containing the 5 ′ end is the second strand of cDNA.
[4] The method according to [3], wherein the cDNA is a cDNA synthesized using, as a template, mRNA in which a polynucleotide having substantially the same base sequence as the oligonucleotide is added to the 5 ′ end.
[5] The method according to [3], which comprises a step of synthesizing a polynucleotide having an unknown base sequence in the region containing the 5 ′ end by incubating the next element under conditions that allow complementary strand synthesis.
(i) first strand of cDNA
(ii) an enzyme that catalyzes a template-dependent complementary strand synthesis reaction
(iii) an enzyme having 5'-3 'exonuclease activity, and
(iv) Nucleotide substrate
[6] The method according to [5], wherein (i) an enzyme having 5′-3 ′ exonuclease activity is used as an enzyme that catalyzes a template-dependent complementary strand synthesis reaction.
[7] The method according to [5], wherein (i) an enzyme having reverse transcriptase activity is used as an enzyme that catalyzes a template-dependent complementary strand synthesis reaction.
[8] The method according to [5], comprising a step of adding an oligonucleotide having a palindromic structure to 3 ′ of the first strand of cDNA prior to incubation.
[9] The method according to [1], wherein the polynucleotide whose base sequence in the region including the 5 ′ end is unknown is the first strand of cDNA.
[10] The method according to [9], wherein the polynucleotide is cDNA, and the cDNA is cDNA synthesized by an oligo dT primer having an arbitrary base sequence added to 5 ′.
[11] A polynucleotide having an unknown terminal base sequence, comprising a step of cloning a polynucleotide having an unknown base sequence in the region including the 5 ′ end synthesized according to the method of [3] or [9] Isolation method.
[12] A cDNA library comprising a cDNA having a palindromic structure at the 3 ′ end of the first strand and / or the second strand of cDNA.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for synthesizing a polynucleotide having an unknown base sequence in a region including the 5 ′ end, comprising the following steps.
a) Step of synthesizing a complementary strand using an oligonucleotide that anneals to an arbitrary region of the polynucleotide as a primer
b) adding a base sequence complementary to the oligonucleotide of step a) to the 3 ′ end of the complementary strand synthesized in step a); and
c) using the complementary strand obtained in step b) as a template, synthesizing a polynucleotide whose base sequence of the region including the 5 ′ end is unknown using the oligonucleotide of step a) as a primer,
[0020]
In the present invention, a polynucleotide whose base sequence in the region including the 5 ′ end is unknown includes any polynucleotide that can be used as a template for complementary strand synthesis. Specifically, DNA, RNA, or hybrid polynucleotides thereof, and polynucleotides including various nucleotide derivatives can be shown. The polynucleotide may be not only naturally derived but also an artificially produced polynucleotide. Alternatively, a polynucleotide in which an artificial mutation is introduced into a naturally occurring polynucleotide can be used.
In the present invention, particularly important polynucleotides are mRNA and / or cDNA. The cDNA here includes both the first strand and the second strand. The region including the 5 ′ end of cDNA and cDNA synthesized using mRNA as a template is an important polynucleotide that often requires base sequence determination.
[0021]
In the present invention, the phrase “the base sequence of the region containing the 5 ′ end is unknown” means that the base sequence of the region containing the 5 ′ end of the polynucleotide used as the template for complementary strand synthesis in step a) is unknown. Therefore, if the polynucleotide is generated using another polynucleotide X as a template, the base sequence on the 3 ′ side of the polynucleotide X is unknown.
When the polynucleotide X is mRNA, the 3 ′ end is usually poly (A). In the present invention, “the region including the 3 ′ end of mRNA is unknown” refers to the case where the base sequence of the region excluding the poly (A) tail at the 3 ′ end is unknown. Furthermore, in cDNA synthesized using the region containing the 3 ′ end of the mRNA as a template, the t of the region containing the 5 ′ end is present regardless of the presence or absence of t stretch (t stretch) located at the 5 ′ end. When the base sequence excluding the linkage is unknown, the base sequence at the 5 ′ end of the polynucleotide is said to be unknown.
However, in step b), the 3 ′ end to which a predetermined base sequence should be added may be the 3 ′ end of the complementary strand generated using the t chain as a template. That is, poly (A) is not considered in determining whether it is unknown. On the other hand, when a base sequence is added to the 3 ′ end, if there is a chain a at the 3 ′ end, the base sequence can be added to the 3 ′ end of the chain a.
[0022]
In the present invention, step a) the step of synthesizing a complementary strand using an oligonucleotide that anneals to an arbitrary region of the polynucleotide as a primer may be performed by any enzyme that catalyzes a primer-dependent complementary strand synthesis reaction. it can. An example of such an enzyme is Taq polymerase.
[0023]
On the other hand, the oligonucleotide used as a primer in step a) anneals to any region of the polynucleotide to prime complementary strand synthesis, and enables the synthesis of the complementary strand to the region reaching the 5 ′ end of the polynucleotide. If it is a thing, the base sequence and length will not be restrict | limited. Generally, in order to start a complementary strand synthesis reaction while maintaining a certain degree of specificity, an oligonucleotide consisting of at least 15 bases, usually 20 to 40 bases, for example 20 to 30 bases, is used as a primer. Methods for preparing such oligonucleotides are known.
[0024]
Next, the base sequence constituting the oligonucleotide is composed of a base sequence complementary to an arbitrary region of the polynucleotide. In the present invention, the term “complementary” refers to a base sequence that can form a base pair (complementary strand bond) according to the Watson-Crick rule. The base sequence of the oligonucleotide of the present invention may not be completely complementary. That is, an oligonucleotide capable of initiating complementary strand synthesis under stringency capable of maintaining specificity is included in the oligonucleotide of the present invention even if its base sequence is not completely complementary. For example, it is an important condition that the base at the 3 ′ end of the primer is complementary to the template. However, the middle part and 5 ′ end of the primer need not be completely complementary.
[0025]
In the present invention, the nucleotide sequence of the region containing the 5 ′ end of the polynucleotide is unknown. However, in other regions, there are parts where the base sequence is clear. Therefore, the oligonucleotide of the present invention can be designed so as to contain a base sequence complementary to a portion where the base sequence is clear. If the polynucleotide is cDNA, it is designed so that a complementary strand synthesis reaction directed to a region whose base sequence is unknown can be primed by annealing to a portion where the base sequence is clear in the base sequence constituting the gene.
[0026]
The present invention includes a step of adding a base sequence complementary to the oligonucleotide of step a) to the 3 ′ end of the complementary strand synthesized in step b) step a) following step a). Step b) can be carried out by any method. As step b) in the present invention, for example, the following method can be shown. That is, step b) can be carried out by incubating the following elements under conditions that allow complementary strand synthesis reaction.
(1) Enzyme that catalyzes template-dependent complementary strand synthesis reaction
(2) the polynucleotide synthesized in step a), and
(3) Nucleotide substrate
[0027]
(1) If an enzyme having 5'-3 'exonuclease activity is used as an enzyme that catalyzes a template-dependent complementary strand synthesis reaction, the element (1) and (2) can be combined with a single enzyme. This is desirable. Examples of such enzymes include Taq DNA polymerase (manufactured by Boehringer Mannheim) ExTaq DNA polymerase, TAKARA Taq (both manufactured by Takara Shuzo), AmpliTaq (manufactured by Applied Biosystems), Tfl, and Tth (both manufactured by Promega). .
[0028]
Or when using DNA polymerase which does not have 5'-3 'exonuclease activity, the other enzyme which has 5'-3' exonuclease activity can be added additionally. For example, λexo (λphage) has double-strand specific 5′-3 ′ exonuclease activity. In the above step, a nucleotide substrate that can be used as a substrate by an enzyme that catalyzes a template-dependent complementary strand synthesis reaction is added. Specifically, nucleotide substrates generally used in PCR methods or cDNA synthesis can be used. Usually, natural deoxynucleotides of atc or g are used. In addition, nucleotide derivatives modified with fluorescent molecules, radioisotopes, biotin, or the like can be used as necessary.
[0029]
By incubating these elements together with the polynucleotide synthesized in (3) step a) under conditions allowing a complementary strand synthesis reaction, at the 3 ′ end of the polynucleotide synthesized in step a), A base sequence complementary to the base sequence can be added. No reaction has been reported so far to add the target nucleotide sequence to the 3 ′ end of DNA by incubating the above elements. The present inventors can add the target base sequence to the end of the polynucleotide under the specific conditions as described above, and further use the added base sequence to make the end base sequence unknown. The present invention was completed by discovering that polynucleotides can be synthesized.
[0030]
The addition of a base sequence to the 3 ′ end of the polynucleotide by incubation of elements (1)-(3) can be explained by the following mechanism (FIG. 4). (3) The polynucleotide synthesized in step a) forms a double strand with the template polynucleotide. Linear double-stranded DNA is circular, especially at low concentrations, and both ends are close together (Heffron et al., In vitro mutagenesis of circular DNA molecule by using synthetic restriction site. Proc. Natl. Acad. Sci. USA 75: 6012-6016,1978). It is believed that DNA in solution is physically stabilized by taking a circular form.
[0031]
If the DNA is at a sufficiently stable temperature, the double strand is stabilized. However, for example, under conditions where a complementary strand synthesis reaction is performed using a heat-resistant DNA polymerase, the substrate is placed under a considerably high temperature. For example, the complementary strand synthesis reaction by Ex-Taq (Takara Shuzo) uses a high temperature of around 70 ° C. Under such a high temperature condition, the terminal portion of the double-stranded DNA cannot stably maintain the double-stranded structure. As a result, a reaction may occur in which the 3 ′ end approaches a strand other than the strand to be originally base paired, and further, complementary strand synthesis is initiated using that strand as a template. In the present invention, this phenomenon is called template switching of DNA polymerase.
[0032]
At this time, the strands that are most likely to undergo cross-linkage are both ends of the linear DNA that are approached by taking a circular structure. More specifically, the 3 ′ end approaches the 5 ′ end of the same strand, and a base sequence complementary to the base sequence is synthesized. As a result, a complementary base sequence is added to the 5 ′ end of the 3 ′ end. Since its own 5 ′ end is the base sequence of the oligonucleotide that is originally a primer, as a result, a complementary sequence of the oligonucleotide is added to the 3 ′ end.
[0033]
In the present invention, the mechanism for adding the target base sequence to the 3 ′ end of the polynucleotide is not limited to the mechanism described here. In any case, step b) is achieved by incubating under the conditions described herein. This is supported by the results of the examples.
[0034]
In the present invention, when cDNA is used as the polynucleotide, either the second strand or the first strand can be used as the cDNA. The first strand of cDNA is DNA synthesized using mRNA as a template. The second strand refers to DNA synthesized using the first strand of cDNA as a template. When using the second strand as a template, 5'RACE can be realized because DNA synthesis proceeds in the 5 'direction of mRNA. Conversely, when the first strand is used as a template, DNA synthesis proceeds to the 3 ′ side of the mRNA, so 3 ′ RACE can be performed. In order to use the second strand as a template, a primer having a base sequence complementary to the second strand is designed. If the first strand is a template, the primer base sequence is a base sequence complementary to the first strand. The 5 ′ side of the second strand of cDNA (ie, 5 ′ side of mRNA) contains a translation initiation codon. Therefore, the region on the 5 ′ side of the second strand is important in estimating the translated amino acid sequence based on the cDNA base sequence. Therefore, determining the base sequence of this region is often an important issue in gene isolation and functional analysis.
[0035]
When the second strand of cDNA is used as the polynucleotide in the present invention, the second strand can be synthesized by incubating the following elements (i)-(iv) under conditions that allow complementary strand synthesis.
(i) first strand of cDNA
(ii) an enzyme that catalyzes a template-dependent complementary strand synthesis reaction
(iii) an enzyme having 5'-3 'exonuclease activity, and
(iv) Nucleotide substrate
[0036]
In the present invention, (i) the first strand of cDNA can be synthesized by an enzyme having reverse transcriptase activity using an oligonucleotide having a base sequence complementary to an arbitrary region of mRNA as a primer. The primer for synthesizing the first strand is preferably an oligonucleotide having the same base sequence as the oligonucleotide used in step a). In a general cDNA synthesis method, the mRNA used as a template after the first strand is synthesized is degraded. mRNA can be easily degraded by enzymatic action such as alkali denaturation or RNAseH. When mRNA is degraded, the first strand of cDNA becomes a single strand. In the present invention, an enzyme that catalyzes a template-dependent complementary strand synthesis reaction used in the synthesis of the second strand or step b) can also be used as the reverse transcriptase for the synthesis of the first strand. A DNA polymerase having a reverse transcriptase activity, such as Tfl or Tth, is known. These DNA polymerases are commercially available (Promega). By using such an enzyme, the number of enzymes constituting the reaction system can be reduced.
[0037]
The second strand of cDNA is DNA synthesized using the first strand thus synthesized as a template. The template-dependent complementary strand synthesis reaction usually uses a complementary strand synthesis reaction from the 3 ′ end of the primer. The base sequence to be synthesized can be specifically synthesized by annealing specific to the base sequence of the primer. However, the primer is not an essential condition for complementary strand synthesis. For example, when the above elements (i)-(iv) are incubated under conditions that allow complementary strand synthesis, a complementary strand synthesis reaction using the first strand of cDNA as a template is achieved. The conditions under which complementary strand synthesis is possible here refer to conditions under which the enzyme catalyzing the element (ii) template-dependent complementary strand synthesis reaction can maintain its activity. Those skilled in the art can appropriately set the conditions necessary for maintaining the enzyme activity according to the enzyme used.
[0038]
The synthesis mechanism of the cDNA second strand by incubation of the above elements (i)-(iv) can be explained, for example, as follows. First, the 3 ′ end of the first strand of cDNA approaches itself and initiates a complementary strand synthesis reaction using itself as a template. The 3 ′ end of the first strand does not necessarily have a self-complementary base sequence. However, even if there are several bases, if a complementary strand synthesis reaction occurs using itself as a template, the 3 ′ end acquires a self-complementary base sequence. As a result, base pairing to itself is further stabilized, and eventually the 3 ′ end takes a complete stem-loop structure. If a stem-loop structure can be realized, a complementary strand synthesis reaction using itself as a template proceeds and the synthesis of the second strand of cDNA is completed.
[0039]
The phenomenon of initiating a complementary chain synthesis reaction using itself as a template by the proximity of the 3 ′ end to itself has also been observed in Tetrahymena, for example. Alternatively, the complementary strand synthesis reaction using itself as a template from the 3 ′ end of the first strand is also used in cDNA synthesis (Maniatis T. “Enzymic in vitro synthesis of globin genes” Cell 7: 279-288, 1976).
Therefore, by incubating the above elements (i)-(iv), it is possible to initiate a complementary strand synthesis reaction using itself as a template. The reaction for actually synthesizing the second strand of cDNA under these conditions is described in the Examples.
[0040]
Now, based on such a mechanism, the completion of the second strand results in the synthesis of cDNA in which the first strand and the second strand are continuous on the same strand. Such a cDNA has a double-stranded part annealed with both and a hinge part constituting a stem loop. Here, the enzyme having the element (iii) 5′-3 ′ exonuclease activity digests the first strand constituting the double-stranded portion of the cDNA from the 5 ′ end. Since digestion of cDNA acts on double-stranded DNA, the first strand is digested throughout its entire length leading to the hinge part. Thus, a single-stranded second strand is finally generated. The generated second strand has an unknown base sequence including the 5 ′ end and has a base sequence complementary to the primer used for the synthesis of the first strand at the 3 ′ end. It can be used as a nucleotide.
[0041]
As a step of adding a base sequence complementary to the oligonucleotide of step a) to the 3 ′ end of the complementary strand synthesized in step b) step a) in the present invention, the following method can also be shown. That is, the first strand of cDNA is synthesized using as an template mRNA with a polynucleotide having substantially the same base sequence as that of the oligonucleotide at the 5 ′ end. A base sequence complementary to the base sequence of the oligonucleotide is arranged at the 3 ′ end of the first strand thus synthesized. That is, this cDNA first strand is nothing but the target polynucleotide of step b).
[0042]
A method for linking an arbitrary oligonucleotide to the 5 ′ end of mRNA is known. For example, the oligo cap method, which is a synthesis method of full-length cDNA, is a method for specifically binding an oligonucleotide called an oligo cap linker to the 5 ′ end of mRNA (Murayama, K. and Sugano, S. ( 1994) Oligo-capping: a simple method to replace the cap structure of eucaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174.). In the oligo-cap method, a linker is bound to a capping structure at the 5 ′ end of eukaryotic mRNA. Therefore, contamination of cDNA derived from incomplete mRNA having no capping structure at the 5 ′ end can be prevented.
[0043]
In order to apply the oligo cap method to the present invention, the base sequence of the oligo cap linker may be substantially the same as that of the oligonucleotide. Usually, in the oligo cap method, it is advantageous to use a base sequence that is difficult to find in a gene so that the full-length cDNA is specifically amplified. This is because when a base sequence found in a gene is used, the possibility that a fragment of the gene is amplified instead of the full-length cDNA cannot be denied. On the other hand, in the present invention, since a base sequence complementary to the oligonucleotide is introduced at the 3 ″ end, the base sequence of the linker is rather the base sequence found in the gene. In the present invention, an oligonucleotide is added to the 5 ′ end of mRNA by the same operation as in the oligo cap method except that the base sequence of the linker is different.
[0044]
In the present invention, the first strand of cDNA can be used as the polynucleotide whose base sequence in the region including the 5 ′ end is unknown. The first strand of cDNA is usually synthesized using an oligo dT primer. Next, the second strand is synthesized using the first strand as a template using an oligonucleotide for a region in which the base sequence is clear in the gene. As a result, the 3 ′ end of the second strand is composed of a continuous base sequence of a. The same base sequence is unsuitable for step b) by incubating said elements (1)-(3). The base sequence of the oligonucleotide constituting the 5 ′ end of the second strand is not usually a monotonous base sequence such as a t chain. Therefore, the step b) due to the incubation of the elements (1) to (3) described above is less likely to occur. Such inconvenience can be avoided by adding an appropriate base sequence to the base sequence at the 3 ′ end of the second strand.
[0045]
For example, an arbitrary base sequence can be given to the 3 ′ end of the second strand by adding an appropriate base sequence to the 5 ′ end of the oligo dT primer. For example, a 6-8 base restriction enzyme recognition sequence can be added to the 5 'end of an oligo dT primer. Such an oligo dT primer enables addition of an appropriate base sequence to its 3 ′ end and is useful for ligation of cDNA to a cloning vector. Alternatively, the step b) can be performed by adding substantially the same base sequence as the oligonucleotide to the 5 ′ end of the oligo dT primer.
[0046]
In the present invention, prior to the incubation of the elements (i) to (iv), an oligonucleotide having a palindromic structure at the 5 ′ end and / or 3 ′ end of a polynucleotide whose region sequence including the 5 ′ end is unknown Can be added. The palindromic structure refers to a base sequence including a self-complementary sequence that can form a hairpin loop at the end. The number of bases constituting the self-complementary sequence is not limited. More specifically, a self-complementary sequence of 2 to 20 bases, for example 2 to 4 bases can be shown. Specifically, 5′-aagctt-3 ′ can be used as the palindromic structure of the present invention.
[0047]
Such an additional base sequence can also be realized, for example, by adding a self-complementary sequence to the 5 ′ end of the oligonucleotide. At this time, the addition of a self-complementary sequence and the addition of a restriction enzyme site to the 5 ′ end of the primer can also be performed. For example, the base sequence 5′-aagctt-3 ′ exemplified above is one of the restriction enzyme recognition sequences. A palindromic structure can also be added to the 3 ′ end of the polynucleotide. For example, in synthesizing the polynucleotide, a complementary sequence of the base sequence to be added may be added to the 5 ′ end of the template polynucleotide. For example, a reaction for adding an artificial base sequence to the 5 ′ end of mRNA as in the oligo cap method described above is known. By utilizing this reaction, a self-complementary sequence necessary for the palindromic structure can be added to the 5 ′ end of mRNA. The cDNA synthesized using the mRNA into which the target base sequence is introduced as a template has a palindromic structure at its 3 ′ end.
[0048]
In any case, whether the second strand or the first strand, a polynucleotide having a base sequence complementary to the oligonucleotide added to the 3 ′ end of the region where the base sequence is unknown by the reaction as described above It can be.
The assembly of polynucleotides obtained by adding a palindromic structure to the terminal thus obtained is useful as a cDNA library for the polynucleotide synthesis method according to the present invention. By using this cDNA library, both 5'RACE and 3'RACE can be performed using a single gene-specific primer. That is, 5′RACE can be achieved by a primer that anneals to the second strand of cDNA, and 3′RACE can be achieved by a primer that anneals to the first strand. If a cDNA library composed of cDNAs having palindromic structures at the ends is constructed, unknown base sequences of various genes can be easily obtained based on the present invention using the same library. In the method for synthesizing a polynucleotide of the present invention, it is not essential to add a palindromic structure to the 3 ′ end. However, the addition of a palindromic sequence is effective in order to promote the complementary strand synthesis (self-prime) using the gene-specific primer extension product as a template.
[0049]
By using the cDNA library of the present invention, cDNA can be isolated based on the base sequence of an unknown gene identified by the genome project. Once the cDNA is cloned, it is possible to analyze the protein based on the cDNA. The cDNA library of the present invention can be used for isolating splicing variants of known genes with low expression levels. If cDNA libraries derived from various tissues are synthesized based on the present invention, the utility value is high in isolating genes and splicing variants.
[0050]
The polynucleotide having a palindromic structure added to the end can be amplified by the polynucleotide synthesis method using the oligonucleotide as a primer in step c). A method for synthesizing a polynucleotide having a target base sequence using an oligonucleotide having a base sequence complementary to the terminal region of the base sequence to be amplified as a primer is known.
[0051]
In the present invention, step c) can be performed, for example, by PCR. Specifically, first, the polynucleotide obtained in step b) is denatured into a single strand. In the PCR method, the template polynucleotide is generally denatured by heating. The denatured polynucleotide is incubated with the oligonucleotide serving as a primer under conditions that allow complementary strand synthesis from this primer. A person skilled in the art can set conditions under which complementary strand synthesis is possible according to the base sequence of the primer and the composition of the reaction solution. By repeating the denaturation and the complementary strand synthesis reaction, the target polynucleotide can be continuously generated based on the PCR method.
[0052]
For DNA polymerase catalyzing complementary strand synthesis, it is advantageous to use a thermostable enzyme that does not lose its activity even at the denaturation temperature of the polynucleotide. If the previously used (1) enzyme that catalyzes the template-dependent complementary strand synthesis reaction, or (ii) the DNA polymerase used as the enzyme that catalyzes the template-dependent complementary strand synthesis reaction, has heat resistance It can also be used in step c) as it is.
The polynucleotide synthesized in step c) can be separated by a known method such as ethanol precipitation. Alternatively, a polynucleotide of a desired size can be fractionated using a technique such as electrophoresis.
[0053]
Of the known RACE methods, the RACE method put into practical use under the trade name Marathon-ready cDNA has already been described as applying a method called suppression PCR. On the other hand, in the RACE method based on the present invention, base sequences complementary to each other are arranged at both ends of the cDNA. That is, the cDNA finally used as a template in the RACE according to the present invention has an inverted repeat. Such a structure of cDNA can be said to be a structure in which synthesis should be suppressed in the Marathon-ready cDNA by the principle of suppression PCR. However, in practice, the RACE method based on the present invention is possible, and the synthesis of the target cDNA is carried out efficiently as described in the Examples.
[0054]
In the present invention, the following mechanism can be considered as a reason why the target cDNA can be synthesized despite the fact that the cDNA has an inverted repeat.
As described above, the reason why inverted repeats are difficult to use as templates in suppression PCR is that hybridization of complementary sequences on the same strand competes with primer annealing. However, when the base sequence derived from the gene is sufficiently long, hybridization in the same strand is unlikely to occur. As a result, there arises a situation where primer annealing cannot be sufficiently inhibited, that is, suppression PCR does not act. In RACE, a gene having a long base sequence is likely to be a cDNA to be obtained. Therefore, a mechanism for selectively synthesizing a longer base sequence can be realized by using a cDNA having an inverted repeat sequence as a template.
[0055]
In addition, the ratio of the template cDNA to the primer is also an important factor. That is, by using the primer in a large excess with respect to cDNA, the chance of annealing with the primer can be made larger than the hybridization of complementary strands on the same DNA molecule (intra DNA molecule). Specifically, it is advantageous to use a primer having a molar ratio of about 5 to 20 times, preferably 10 times or more, with respect to the expected amount of template. For example, 1 μg of 1 kb double-stranded cDNA (about 9.12 × 10 ¹¹ 10pmol (about 78ng: about 5.9x10) ¹² molecules) to 20 pmol of 24mer primer.
[0056]
The method for synthesizing a polynucleotide according to the present invention is useful for cloning a polynucleotide whose base sequence in the region including the 5 ′ end is unknown. That is, the present invention includes the step of cloning a polynucleotide having an unknown base sequence in the region containing the 5 ′ end synthesized based on the method described in the present invention, and isolating the polynucleotide having an unknown end base sequence Regarding the method.
Methods for cloning synthesized polynucleotides are known. In general, the synthesized polynucleotide can be incorporated into an appropriate cloning vector and cloned. When a restriction enzyme recognition sequence for cloning is added to a primer used for polynucleotide synthesis, the synthesized polynucleotide can be easily inserted into a cloning vector. Alternatively, the synthesized polynucleotide can be ligated to a cloning vector with blunt ends.
[0057]
The base sequence of the cloned polynucleotide can be determined. The base sequence of the inserted polynucleotide is determined using a primer having a base sequence complementary to the base sequence of the vector. For example, if the base sequence of the region containing the 5 'end of the second strand is unknown, 5'RACE is unknown, or the base sequence of the region containing the 5' end of the first strand is unknown. Can be 3'RACE.
[0058]
The use of the polynucleotide synthesis method of the present invention is not limited to RACE. For example, telomerase activity can be measured based on the method of the present invention. Telomerase uses a DNA having a specific base sequence as a substrate, and adds a base sequence consisting of repetition of a specific base sequence called telomeric repeat to its 3 ′ end. Telomere repeats are composed of a base sequence determined for each species. For example, the human telomeric repeat is (TTAGGG) n. The activity of telomerase can be evaluated based on the length of the added telomeric repeat. In order to measure the activity of telomerase according to the present invention, telomerase is allowed to act on the substrate DNA, and then a polymerase having 5′-3 ′ exonuclease activity is allowed to act. Based on the mechanism described above, the 3 ′ end of the substrate DNA anneals to itself to synthesize a complementary strand, and the extension product of the substrate DNA that has become the template itself is digested by the 5′-3 ′ exonuclease activity. Is done.
[0059]
Since the complementary strand synthesized here has a base sequence complementary to the substrate DNA, complementary strand synthesis is possible using an oligonucleotide having the same base sequence as that of the substrate DNA as a primer (step a)). The substrate DNA remaining in the reaction solution can also be used as a primer as it is. When complementary strand synthesis reaches the 5 'end, a base sequence complementary to the substrate DNA is added to the 3' end by circularization of double-stranded DNA and template switching action (step b)). Finally, by using an oligonucleotide having the same base sequence as that of the substrate DNA, the unknown base sequence on the 3 ′ end side of the substrate DNA extended by the action of telomerase can be amplified.
[0060]
It is clear that the base sequence added by the action of telomerase is telomeric repeat. However, it is unclear how long the telomeric repeat at the end and the terminal telomere repeat has. Therefore, DNA synthesized using a substrate DNA extended by the action of telomerase as a template is included in a polynucleotide whose base sequence of the region including the 5 ′ end in the present invention is unknown.
[0061]
By combining elements necessary for the polynucleotide synthesis method of the present invention, a polynucleotide synthesis kit containing an unknown base sequence can be obtained. The kit based on this invention can be comprised by the following element, for example.
-DNA polymerase having 5'-3 'exonuclease activity
-A buffer capable of maintaining the activity of said DNA polymerase; and
-Nucleotide substrates for complementary strand synthesis
[0062]
The specific configuration of each element in the present invention is as described above. In addition to the above elements, additional elements can be added to the kit of the present invention. For example, a reverse transcriptase for synthesizing the first strand of cDNA and an oligo dT primer for performing 3′RACE can be combined. Alternatively, an oligonucleotide primer designed for a region where the nucleotide sequence of the target polynucleotide is clear can be attached in advance.
[0063]
In addition, when a linker is introduced at the 5 ′ end of mRNA, an oligonucleotide or RNA ligase used as a linker can be combined. Furthermore, when the oligocap method is applied to the linker bond to the 5 ′ end of mRNA, various enzymes necessary for the oligocap method are combined. For example, alkaline phosphatase (BAP) or tobacco acid pyrophosphatase (TAP) can be combined.
[0064]
The kit of the present invention can be combined with controls to determine if the experimental materials and procedures were appropriate. For the control in the present invention, for example, a primer that can be expected to synthesize a cDNA of a certain size can be used in any cDNA library. As a primer that gives such cDNA, for example, a primer that enables synthesis of β-actin gene can be shown.
Hereinafter, the present invention will be described more specifically based on examples.
[0065]
【Example】
Comparative example. Known RACE method and RACE method of the present invention
As a known RACE method, Marathon cDNA amplification kit (manufactured by Clontech) was used to try to isolate an unknown nucleotide sequence on the 5 ′ side of the gene. Mouse Msh4 was selected as the gene. Msh4 is a member of the MutS mismatch repair gene family and has been identified in S. cerevisiae, C. elegans, mice and humans (Ross-Macdonald and Roeder, Cell 79: 1069-80 (1994); Zalevsky et al. , Genetics 153: 1271-83 (1999); Kneitz et al., Genes Dev. 14: 1085-97 (2000); Paquis-Flucklinger et al., Genomics 44: 188-94 (1997)) The base sequence of Msh4 is Although it is already known, the purpose of this time was to obtain the splicing variant. MRNA was extracted from the testes of mice (8 weeks old), and cDNA was synthesized according to a conventional method. Next, adapters were ligated to both ends of the cDNA. The above process was in accordance with the Clontech manual. PCR was performed using the obtained adapter-added cDNA as a template and primers specific to mouse Msh4 splicing variant γ (Msh4-γ) and β-actin. The reaction sequence and the composition of the reaction solution are shown below.
[94 ° C / 30 seconds → 60 ° C / 1 minute → 68 ° C / 3 minutes] x 35 cycles
2μL 10x reaction buffer
1μL 2.5mM 4dNTPs
1μL testicular cDNA (with adapter)
0.2μL Ex-Taq DNA polymerase
0.4μL each gene specific primer (10μM)
0.4μL adapter primer (10μM)
15μL distilled water (total 20μL)
The base sequences of the primers used in the experiment are as follows. # 18 and # 20 are gene-specific primers (GSP) designed based on the base sequence of mouse Msh4.
# 18: 5'-GGCATAGTTCCAGGAAATGGGTAG-3 '(SEQ ID NO: 1)
# 20: 5'-GGTATTGTTGGCGACAGGTTTCTC-3 '(SEQ ID NO: 2)
β-actin 1: 5'-GTGACGAGGCCCAGAGCAAGAG-3 '(SEQ ID NO: 3)
β-actin 2: 5'-AGGGGCCGGACTCATCGTACTC-3 '(SEQ ID NO: 4)
A part of the reaction solution after the reaction was taken and electrophoresed on an agarose gel. DNA in the agarose gel was detected by ethidium bromide staining. The result of Msh4 is shown in FIG. 2, and the result of β-actin is shown in FIG.
[0066]
In the Marathon cDNA amplification kit, as shown in FIG. 1, the adapter primer is designed to have the same sequence as the 5 ′ overhanging single-stranded portion of the adapter. The 3 'end of the oligonucleotide constituting the adapter is NH ₂ Since it is blocked by a group, synthesis of complementary strands starting from this is inhibited. Therefore, in principle, complementary strand synthesis from an adapter primer does not occur unless complementary strand synthesis from a gene specific primer (GSP) occurs.
[0067]
However, in practice, a non-specific product was synthesized and a smear background appeared (FIG. 2, FIG. 2, lane 1). These smear bands that seemed to be products of non-specific reactions were thought to have been generated due to the base sequence of the adapter primer and other phenomena. The Clontech manual points out the possibility of the following non-specific reactions. For example, it is described that a complementary strand is synthesized in an overhang portion of an adapter linked to cDNA, and cDNA may be amplified without synthesizing a complementary strand from a gene-specific primer.
[0068]
Therefore, in order to suppress the synthesis of the product resulting from the non-specific reaction as described above, PCR was attempted by limiting the amount of the adapter primer. That is, PCR was performed under the conditions that the amount of adapter primer used for ligation with cDNA was 1/10 to 1/1000, and no adapter primer was used. As a result, a background was observed at 1/10 (lane 2 in FIGS. 2 and 3). When it was restricted to 1/100 to 1/1000, synthesis of non-specific products was hardly observed (lanes 3 to 4 in FIGS. 2 and 3). An amplification product was observed (lane 5 in Fig. 3), that is, the amplification product was also detected by PCR using only gene-specific primers.
[0069]
As a result of analyzing the base sequence of the synthesized PCR product, it was confirmed that the target DNA was synthesized in all but one. Based on these results, it was shown that a cDNA having an unknown structure that can be amplified with only a gene-specific primer without using an adapter primer was synthesized.
[0070]
For example, it was considered that the mechanism shown in FIG. 4 made it possible to synthesize DNA without using an adapter primer.
Cycle 1:
Similar to normal PCR, double-stranded DNA is synthesized from gene specific primers.
Cycle 2:
At a certain frequency, the vicinity of the 3 ′ end of the single strand forms a hairpin structure, and double-stranded DNA is synthesized using this portion as a primer and itself as a template. DNA replication using a hairpin structure as a primer is a phenomenon observed in the synthesis of Tetrahymena rDNA. Most of the synthesized double-stranded DNA is degraded into single strands by the double-stranded DNA-specific 5′-3 ′ exonuclease activity of Taq DNA polymerase. At this time, if it is not affected by the 5′-3 ′ exonuclease activity, DNA having the structure shown by cycle 3-no exo attacked is generated. However, it is unlikely that DNA having such a structure is synthesized.
[0071]
Cycle 3:
Double-stranded DNA is synthesized using the single-stranded DNA generated in cycle 2 as a template. Since DNA synthesis is performed at a high temperature of 68 ° C., a part of the previously synthesized double-stranded ends cannot maintain a double-stranded structure. As a result, Taq DNA polymerase, which has been synthesized up to the 5 ′ end of the template, continues to synthesize using a new complementary strand synthesized by itself as a template, and also synthesizes DNA complementary to the primer base sequence. It has already been mentioned that such a reaction is called template switching action. A base sequence complementary to the primer base sequence is located at the 3 ′ end of the newly synthesized complementary strand.
Cycle 4:
In subsequent cycles, DNA is amplified as in normal PCR. However, since the complementary sequence of the primer is always located at the 3 ′ end of the DNA, only one kind of primer is necessary for PCR.
[0072]
Example
As shown in FIG. 4, the following experiment was performed in order to confirm that an unknown base sequence can be amplified without using an adapter. That is, 5′RACE was performed using only the gene-specific primer for the same cDNA as in the above comparative example or a cDNA library derived from E12.5 mouse fetal brain. DNA was synthesized under the same conditions as in the above Comparative Example except that the step of ligating the adapter to cDNA was omitted and no adapter primer was added in PCR. The base sequence of the gene-specific primer used in the experiment is shown below.
# 6: 5'-GTGGACTTTCCGCTCATATTTGGT-3 '(SEQ ID NO: 5)
# 18: 5'-GGCATAGTTCCAGGAAATGGGTAG-3 '(SEQ ID NO: 1)
# 20: 5'-GGTATTGTTGGCGACAGGTTTCTC-3 '(SEQ ID NO: 2)
# 21: 5'-GGGATGCCATCCTTGTTTGATTGC-3 '(SEQ ID NO: 6)
# 26: 5'-AAGAGACTGTCAGGCATGGTAGTG-3 '(SEQ ID NO: 7)
# 27: 5'-TAATCCCAGCACTCAGGAGGCAGA-3 '(SEQ ID NO: 8)
β-actin 1: 5'-GTGACGAGGCCCAGAGCAAGAG-3 '(SEQ ID NO: 3)
β-actin 2: 5'-AGGGGCCGGACTCATCGTACTC-3 '(SEQ ID NO: 4)
Among the above primers, # 6, # 20, and # 21 are primers designed based on the base sequence of Msh4-γ. The regions where each primer anneals are arranged in the order of # 6, # 20, and # 21 from the 3 ′ side of the Msh4-γ cDNA. On the other hand, # 26 and # 27 are primers designed based on the base sequence of the Msh4 splicing variant ε (Msh4-ε). The regions where each primer anneals are arranged in the order of # 26 and # 27 from the 3 ′ side of the Msh4-ε cDNA. Therefore, nested PCR can be performed by combining these primers.
[0073]
A part of the results using # 26, # 27, or # 18 as a gene-specific primer is shown in FIG. FIG. 5 shows the results of reacting each primer in triplicate or in triplicate. As a template, a cDNA library derived from mouse fetal brain was used. It was confirmed that the target amplification product was amplified by one kind of primer.
[0074]
Furthermore, the base sequence of the terminal part of the base sequence of the amplified DNA was determined, and it was confirmed that the base sequence complementary to the primer was added to the 3 ′ end. The structure of the determined terminal portion is shown in FIG. 6 and FIG. In the figure, the base sequence surrounded by a square indicates the base sequence derived from the primer, and the underlined portion indicates the complementary base sequence other than the primer. The complementary base sequence in the underlined part confirms that a complementary sequence (inverted repeat) was synthesized by template switching.
[0075]
This experiment confirmed the amplification of cDNA of the following size for each splicing variant of Msh4. According to the present invention, it has been shown that a region containing an unknown base sequence can be amplified over a sufficient length by a single primer. In the experiment, only variants γ and ε were used. All variants synthesized in this experiment contain the same exons as variants γ or ε. Therefore, various variants were synthesized with primers for variants γ and ε. For example, the variants αβθ and ι both contain a base sequence that the # 20 and # 21 primers anneal.
α: about 2 kb
β: about 2 kb and about 1.5 kb
δ: about 1 kb
ε: about 2 kb
θ: about 1.5 kb
ι: About 1.5kb
[0076]
As is apparent from the gel photographs in FIGS. 2 and 3, if the target is present and the PCR conditions are optimal, the cDNA is amplified as a very clean single band by the method of the present invention. The polynucleotide synthesis method according to the present invention is a very unique method and is highly reliable. The high reliability of the method of the present invention is supported by the fact that no abnormalities were found in the terminal nucleotide sequence when compared to the genomic DNA sequence for all Msh4 variants. According to the method of the present invention, it is possible to isolate those having an unusual structure such as αδε that is very little contained in a cDNA library. Using the method of the present invention, it may be possible to construct an unusual cDNA library based on the information of the genome project.
[0077]
【The invention's effect】
The present invention provides a method for synthesizing a polynucleotide whose base sequence in the region containing the 5 ′ end is unknown. When the method of the present invention is applied to the second strand of cDNA, 5′RACE can be realized. The 5′RACE based on the present invention made it possible to obtain a longer 5 ′ cDNA that could not be obtained by a known method. The method of the present invention also allows 3'RACE when applied to the first strand of cDNA. In the present invention, the same principle can be applied to both 5′RACE and 3′RACE.
[0078]
According to the method for synthesizing a polynucleotide of the present invention, a method for synthesizing a polynucleotide whose base sequence of the region including the 5 ′ end is unknown by a simple operation by using a reverse transcriptase, one primer, and a specific DNA polymerase. Is possible. That is, according to the present invention, the target polynucleotide can be synthesized by an inexpensive and simple technique. On the other hand, the known method requires the use of a special primer and has been carried out based on a complex reaction in which a plurality of enzymes are combined.
[0079]
The method of the present invention can efficiently synthesize the target polynucleotide. “Efficient” means that the following characteristics can be expected.
-The unknown base sequence can be obtained in a longer state.
-Suppressing non-specific reactions
The non-specific reaction mentioned here refers to a reaction for synthesizing a polynucleotide other than the target polynucleotide. Specifically, the reaction that generates a primer dimer, a polynucleotide having a structure in which a plurality of cDNA fragments are linked, and the like is included in the non-specific reaction.
Due to this feature, 5′RACE and 3′RACE based on the present invention enable reliable acquisition of the target cDNA.
[0080]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the reaction principle of Marathon cDNA amplification kit used in Examples. In the figure, 1 indicates the annealing position of the adapter primer, and 2 indicates the position where the adapter primer anneals for nested PCR. ds cDNA represents a double-stranded cDNA, and NNT30 represents a primer used for cDNA synthesis.
FIG. 2 is a photograph showing a result of electrophoresis of a PCR product obtained by applying 5′RACE method to mouse Msh4 gene. 1st shows the result when the 5′-side primer is used, and 2ndary shows the result when the 3′-side primer is used.
FIG. 3 is a photograph showing a result of electrophoresis of a PCR product obtained by applying 5′RACE method to mouse β-actin gene.
FIG. 4 is a view showing a mechanism of DNA synthesis without using an adapter primer.
FIG. 5 is a photograph showing the result of electrophoresis of a PCR product without using an adapter primer.
FIG. 6 is a diagram showing the results of determining the base sequence of the terminal portion of DNA amplified according to the present invention. In the figure, the base sequence of the primer is shown enclosed by a square. In addition, the base sequence complementary to that derived from the primer added to the terminal portion is underlined. Each base sequence is described for each splicing variant, and shows the base sequence of the 5 ′ end on the top and the 3 ′ end on the bottom. The name of the splicing variant and the length of the amplification product are shown on the left side of each base sequence.
FIG. 7 is a diagram showing the results of determining the base sequence of the terminal portion of DNA amplified according to the present invention (continuation of FIG. 6).

Claims (11)

A method for synthesizing a polynucleotide having an unknown base sequence of a region including the 5 ′ end, comprising the following steps:
a) A step of synthesizing a complementary strand using as an primer an oligonucleotide that anneals to any one region of the polynucleotide whose nucleotide sequence is arbitrarily selected from known regions
b) adding a nucleotide consisting of a base sequence complementary to the oligonucleotide of step a) to the 3 ′ end of the complementary strand synthesized in step a); and
c) using the complementary strand obtained in step b) as a template, synthesizing a polynucleotide whose base sequence of the region including the 5 ′ end is unknown using the oligonucleotide in step a) as a primer,
Step b) comprising the step of incubating the following elements under conditions allowing a complementary strand synthesis reaction:
(1) Enzymes that catalyze template-dependent complementary strand synthesis reactions
(2) a double-stranded polynucleotide comprising the complementary strand synthesized in step a) and its template, and
(3) Nucleotide substrate   2. The method according to claim 1, wherein the polynucleotide whose base sequence in the region including the 5 ′ end is unknown is the second strand of cDNA. 3. The method according to claim 2 , wherein the cDNA is a cDNA synthesized using an mRNA having a polynucleotide having the same base sequence as the oligonucleotide added to the 5 ′ end as a template. 3. The method according to claim 2 , comprising a step of synthesizing a polynucleotide whose base sequence of the region including the 5 ′ end is unknown by incubating the next element under conditions capable of complementary strand synthesis.
(I) the first strand of cDNA (ii) an enzyme that catalyzes a template-dependent complementary strand synthesis reaction (iii) an enzyme having 5'-3 'exonuclease activity, and (iv) a nucleotide substrate 5. The method according to claim 4 , wherein an enzyme having 5′-3 ′ exonuclease activity is used as an enzyme that catalyzes a template-dependent complementary strand synthesis reaction. 5. The method according to claim 4 , wherein an enzyme having reverse transcriptase activity is used as an enzyme that catalyzes a template-dependent complementary strand synthesis reaction. 5. The method according to claim 4 , comprising a step of adding an oligonucleotide having a palindromic structure to the 3 ′ end of the first strand of the cDNA prior to the incubation.   2. The method according to claim 1, wherein the polynucleotide whose base sequence in the region including the 5 ′ end is unknown is the first strand of cDNA. 9. The method according to claim 8 , wherein the polynucleotide is cDNA, and the cDNA is a cDNA synthesized by an oligo dT primer in which a nucleotide having an arbitrary base sequence is added to the 5 ′ end . 9. Isolation of a polynucleotide whose terminal base sequence is unknown, comprising a step of cloning a polynucleotide whose base sequence is unknown in the region including the 5 ′ end synthesized based on the method according to claim 2 or claim 8. Method.   The method of claim 1, wherein a mold transfer occurs in step b).

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