JPH09234074A - Adapter double-stranded dna and amplification of dna by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for amplifying DNAs by which various DNAs having a long to a short lengths are simultaneously and uniformly amplified at a time and to obtain an adapter double-stranded DNA utilizable for the amplification. SOLUTION: This adapter double-stranded DNA is the one capable of bonding to a DNA to be amplified only at one terminal and various DNAs are simultaneously and uniformly amplified by performing a PCR(polymerase chain reaction) under the following conditions by using the adapter double-stranded DNA: That is, (1) the adapter double-stranded DNA is bonded to the DNA to be amplified; (2) a primer single-stranded DNA having a base sequence of a single- stranded DNA having the terminal, located on the side unbonded to the DNA to be amplified and corresponding to the 5'-terminal among the single-stranded DNAs composing the adapter double-bonded DNA is used; (3) a long-chain DNA amplifying enzyme is used and (4) the PCR cycle has two steps.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for uniformly amplifying a wide variety of DNAs at the same time, from short ones to long ones.

[0002]

2. Description of the Related Art A conventional DNA amplification method is a polymerase chain reaction (Polymerase Chain) using Taq polymerase (Taq polymerase).
Reaction (hereinafter referred to as PCR) has been mainly used. This method uses Ta that is a thermostable DNA replication enzyme.
By using q polymerase, denaturation of DNA, annealing (binding) of primers, and synthesis of DNA (extension reaction) are continuously repeated in a test tube (tube). First, the denaturation of DNA is performed at a temperature of 90 ° C. or higher, and the annealing of the primer is 50 to 7
At a temperature of about 0 ° C, DNA synthesis is performed at about 68 to 75 ° C. Taq po, a thermostable DNA replication enzyme
Lymerase is an enzyme that is not inactivated in these cycles, and it is a Klenow fragment (klenow).
It is possible to continuously repeat denaturation of DNA, annealing (binding) of primers, and synthesis of DNA in a test tube (tube), which has been impossible with the use of fragmentation or the like.

Recently, an enzyme capable of amplifying DNA of 20 kb or more has been developed and used as a thermostable enzyme for PCR. However, PC using this enzyme
The R method is mainly intended to amplify a specific DNA of interest, and it has been difficult to collectively amplify a wide variety of DNAs. On the other hand, some D at a time
Although attempts have been made to simultaneously amplify NA, the number of kinds of DNA to be amplified is as small as about 5 kinds, and the length of the amplified DNA is also about 1 kb. Also,
When DNA is repeatedly amplified, short DNA is easily amplified, but this point is hardly overcome.

[0004]

SUMMARY OF THE INVENTION As described above, a DNA amplification method for uniformly amplifying multiple types of DNA at the same time from a short product to a long product at the same time has not been developed. The object is to develop a DNA amplification method. And the development of an adapter double-stranded DNA preferably used for that purpose. More specifically, PC
In R, the DNA to be amplified (DNA to be amplified) is first hybridized with a primer, but in the case of simultaneously amplifying a wide variety of DNAs from a short one to a long one at the same time, it hybridizes to all DNAs. Since there is no primer, it is an object to develop a DNA amplification method that is not limited to the method of directly hybridizing the primer to the amplified DNA.

[0005]

Therefore, the present inventor has proposed a method of binding an adapter DNA (which is synonymous with an adapter double-stranded DNA) to a DNA to be amplified and using a primer which hybridizes to the adapter DNA, as well as an adapter DNA. And the primer was developed. Furthermore, the present inventors have found that an enzyme used to amplify long-chain DNA (long-chain DNA
Amplification enzyme), reaction temperature, reaction cycle, Mg ²⁺ concentration and other operating conditions and operating methods in PCR were examined. As a result, various types of D using the above-mentioned adapter DNA and primer
A DNA amplification method was developed to simultaneously and uniformly amplify NA.

First, embodiments of the adapter double-stranded DNA of the present invention are listed below. The term “amplified DNA” used in the present specification means the DNA present at the beginning of PCR, and
The adapter binds to NA. Also, the amplified DNA
In the present specification, a DNA in which the DNA and the adapter DNA are bound is referred to as a template DNA. The first aspect of the adapter double-stranded DNA of the present invention satisfies the following requirements. One of the single-stranded DNAs forming the adapter double-stranded DNA is projected at one end so that it cannot bind to the amplified DNA or another adapter double-stranded DNA. Of the single-stranded DNA forming the adapter double-stranded DNA, the single-stranded DNA corresponding to the 5'end is phosphorylated at the end on the side that binds to the amplified DNA. The end of the adapter double-stranded DNA of the first aspect that binds to the DNA to be amplified may be either blunt or have an overhang so that the adapters do not bind to each other and bind to the DNA to be amplified.

The second aspect of the adapter double-stranded DNA of the present invention satisfies the above and below requirements. A restriction enzyme site that produces a sticky end is amplified by D
Included in addition to the end not bound to NA. The third aspect of the adapter double-stranded DNA of the present invention satisfies the above and below requirements. A restriction enzyme site that produces blunt ends is amplified with D
Included in addition to the end not bound to NA. The fourth aspect of the adapter double-stranded DNA of the present invention satisfies the above and the above requirements. The fifth aspect of the adapter double-stranded DNA of the present invention satisfies the above and below requirements. Either or both of EcoRI and NotI restriction enzyme sites are included in addition to the end that does not bind to the DNA to be amplified. Sixth of the adapter double-stranded DNA of the present invention
Aspects satisfy the above and below requirements. An EcoRV restriction enzyme site is included other than both ends on the side not binding to the amplified DNA. . The seventh aspect of the adapter double-stranded DNA of the present invention satisfies the above and below requirements. EcoRI, NotI and EcoRV restriction enzyme sites are included in addition to the end that does not bind to the DNA to be amplified.

The eighth aspect of the adapter double-stranded DNA of the present invention satisfies the following requirements. Single-stranded DNA having the sequence shown in SEQ ID NO: 1 in the sequence listing
And a sequence described in SEQ ID NO: 2 in the sequence listing and hybridized with a single-stranded DNA having a phosphorylated 5 ′ end.

The ninth aspect of the adapter double-stranded DNA of the present invention is a chemical or enzymatic modification of the adapter double-stranded DNA of each of the above aspects.

Next, in the embodiment of the primer single-stranded DNA of the present invention, of the single-stranded DNA forming the adapter double-stranded DNA of the present invention, the end not binding to the DNA to be amplified is 5 ′. It has a single-stranded DNA base sequence corresponding to the end. For example, for the adapter double-stranded DNA of the ninth aspect, one having the base sequence of SEQ ID NO: 1 in the sequence listing is a primer (primer single-stranded DNA
And agree).

Next, aspects of the DNA amplification method of the present invention will be described below. D amplified by the adapter double-stranded DNA of the present invention
Connect with NA. The primer single-stranded DNA of the present invention is used. A long-chain DNA amplification enzyme is used. The PCR cycle is a two step process. The second aspect of the DNA amplification method of the present invention is to carry out a DNA amplification reaction under the above and below conditions. A long-chain DNA amplification enzyme having a high mutation modification ability is used.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The adapter DNA of the present invention can bind to the DNA to be amplified only at one end. Specifically, two single-stranded DNAs forming the adapter DNA
Among them, the single-stranded DNA having the 5'end at the end that binds to the DNA to be amplified (the end on the binding side) is phosphorylated at the end so that the adapter is directly attached to the end of the DNA to be amplified. Can be combined. The end of the adapter DNA on the binding side may be blunt, or may have a protrusion so that the adapters do not bind to each other and can bind to the amplified DNA. The ends of the DNA to be amplified can be blunted with a modifying enzyme as necessary, or the ends can be made to project as restriction enzyme sites by treatment with a restriction enzyme. Should be selected. When synthesizing the amplified DNA, the ends of the DNA can be blunted or can be synthesized. The binding end of the adapter DNA may be selected according to which one is synthesized.

The other end (non-bonding side end) of the adapter is the two single-stranded DNAs forming the adapter DNA.
One of the two is protruding and the amplified DN
Inability to bind to A or other adapter duplex ends. Regarding the overhang, if the ends of the DNA to be amplified are blunt, the non-binding end of the adapter DNA may simply overhang. If the ends of the amplified DNA are not blunt, that is, they are treated with an enzyme, the ends of this DNA serve as restriction enzyme sites.
It suffices that the non-binding end of NA is projected so as not to be a restriction enzyme site. In order to prevent the adapter DNAs from binding to each other, it is sufficient that the protruding single-stranded portion has a sequence that prevents the single strands from hybridizing. The adapter of the present invention makes it possible to attach only one adapter to each end of the amplified DNA in exactly one direction. In addition, it will not happen that adapters are connected to each other, so PC
You can do R. The length of the adapter double-stranded DNA is the length of the amplified single-stranded DNA that composes it.
It is preferable that the protruding length at the end that does not bind to is 16 bases or more. In practice, it is particularly preferable that the single-stranded DNA has 24 bases or more. The upper limit of the length of the single-stranded DNA is not particularly limited, but a length of up to about 40 bases is preferable for practical use.

The adapter DNA of the present invention has, in its base sequence, either or both of a restriction enzyme site which produces a sticky end or a restriction enzyme site which produces a blunt end, or an amplified DNA of the adapter DNA. It is preferable to include it other than the terminal on the side not bound to. PCR
Then, it is treated with the restriction enzyme site, and DNA for the vector is added.
This is because the introduction becomes easy. Blunt ends
When the DNA is introduced into the vector, there is no sequence dependence of the introduced portion, so that the DNA can be easily introduced into any vector. Therefore, it is possible to easily introduce DNA into any vector by the restriction enzyme site that produces a blunt end. The EcoRV site can be preferably used as the restriction enzyme site that produces a blunt end.

[0015] The sticky end improves the efficiency of introducing DNA into the vector several times or more as compared with the blunt end. Therefore,
The restriction enzyme sites that generate sticky ends allow efficient introduction of DNA into any vector. When actually introducing a DNA having a sticky end into a vector, a treatment for aligning the end of the vector with the restriction enzyme site at the end of the DNA to be introduced may be carried out, if necessary. EcoR
The I site is a restriction enzyme site that produces the most versatile sticky ends, and most commercial cDNA library vectors are treated at this site. Therefore, the adapter double-stranded DNA must contain an EcoRI site. As a result, it becomes possible to use a commercially available vector for a cDNA library, and it becomes easy to carry out a DNA operation such as converting the amplified DNA into a cDNA library. Therefore, the EcoRI site can be preferably used. The NotI site is a restriction enzyme that produces a sticky end that recognizes eight bases, and thus has very few cleavage sites. Therefore, the amplified D
When NA is treated with NotI sites, cohesive ends can be created in the DNA with minimal cleavage of internal sequences of the DNA. Therefore, the NotI site can be preferably used as the restriction enzyme site that produces sticky ends.

The restriction enzyme site can be appropriately selected and used depending on the cDNA library vector used other than the above. Further, the number of restriction enzyme sites contained in the adapter DNA is not particularly limited within the range depending on its length. Those having both a restriction enzyme site for producing sticky ends and a restriction enzyme site for producing blunt ends can be preferably used.

The primer single-stranded DNA of the present invention is a single-stranded DNA which constitutes the above-mentioned adapter double-stranded DNA of the present invention.
5'of the end that does not bind to the amplified DNA
Single-stranded D having a base sequence of single-stranded DNA corresponding to the end
NA.

The adapter DNA and primer DNA of the present invention, regardless of their origin, may be those synthesized by, for example, a DNA synthesizer, or may be those extracted from cells or the like and those obtained by processing the extracts. . The length of the primer is preferably 16 bases or more. In practice, it is particularly preferable that the single-stranded DNA has 24 bases or more.

By the way, when chemically synthesizing or enzymatically synthesizing DNA (including processing of natural products), methylation of side chains or biotinylation, or substitution of O in the phosphate group with S. It is well known to modify chemically. Chemical modifications that can be introduced during chemical synthesis include, for example, 1) biotinylation, 2) methylation,
3) Dicoccigenination, 4) Dephosphorylation, 5) Fluorescent labeling (fluorescein, rhodamine, Texas red and its derivatives), 6) Amination, 7) O of S of phosphate group
The main examples are the synthesis of DNA and RNA substituted with. Examples of the enzymatic modification include 1) biotinylation, 2) methylation, 3) dicoxigenation, 4) dephosphorylation, and 5) fluorescent labeling (fluorescein, rhodamine,
Texas red and its derivatives), and 6) enzyme labeling (alkaline phosphatase). Therefore, the adapter DNA of the present invention and the primer DN
A includes the chemically modified or enzymatically modified DNA in its range.

The DNA amplification method of the present invention provides a PCR method under the following conditions, and enables a wide variety of DNAs to be uniformly amplified. In particular, it makes it possible to simultaneously and uniformly amplify a wide variety of DNAs including long DNAs of 1 kb or more, which have been difficult in the past. The adapter DNA of the present invention is ligated with the DNA to be amplified. It is used as the primer DNA of the present invention. A long-chain DNA amplification enzyme is used. The PCR cycle is a two step process.

The long-chain DNA amplification enzyme used in the present invention can be used without particular limitation. Any of the commercially available long-chain DNA amplification enzymes listed in the examples can be preferably used. There are slight differences depending on the manufacturer, and as shown in the examples, Tth XL (registered trademark) manufactured by Perkin Elmer Co., Ltd. can be particularly preferably used. This is considered to be due to the fact that the enzyme is characterized by having a high mutation modification ability, and thus it can be said that an enzyme having a high mutation modification ability can be particularly preferably used.

Regarding the PCR cycle, a two-step cycle of denaturation of DNA at 90 ° C. or higher → annealing at 68 to 75 ° C. and extension reaction may be repeated. The extension reaction time is preferably 10 minutes or more and gradually increased. Further, the above cycle is preferably repeated 28 times or more.

The temperature during the extension reaction may be any temperature at which the DNA synthesis reaction proceeds depending on the enzyme used. The extension reaction is preferably carried out at the optimum reaction temperature. For most enzymes, the optimum reaction temperature is about 70 ° C.

A particularly desirable embodiment of the DNA amplification method of the present invention is to carry out the DNA amplification reaction under the following conditions (1) to (3). As the adapter double-stranded DNA, SEQ ID NO: 1 in the sequence listing
DNA amplified by hybridizing the single-stranded DNA having the sequence described in 1) with the single-stranded DNA having the sequence described in SEQ ID NO: 2 of the sequence listing and having a phosphorylated 5 ′ end thereof Connect with. As the primer single-stranded DNA, SEQ ID NO: 2 in the sequence listing
The single-stranded DNA having the sequence described in 1. is used. Tth XL is used as a DNA amplification enzyme. The PCR reaction was set to hot start, and after reacting at 94 ° C for 1 minute, (94 ° C for 15 seconds → 68 ° C for 1 second
The cycle of 0 minutes) was repeated 16 times, followed by the cycle of (94 ° C. for 15 seconds → 68 ° C. for 11 minutes) 4 times, followed by (94 ° C. for 15 seconds →
A cycle of 68 ° C. for 12 minutes) is repeated 4 times, and then a cycle of (94 ° C. for 15 seconds → 68 ° C. for 13 minutes) is repeated 4 times. The reaction mixture having the following composition was added to the low mix first,
After the sterilized wax has set, up mix1 and up m
ix2 is added and used. low mix (first reaction solution) primer (5 pmol / μl) 1.0 μl Mg (OAc) ₂ (25 mM) 1.2 μl dNTP (2.5 mM) 1.6 μl 3.3 × reaction buffer 2.4 μl sterile distillation Water 1.8 μl Sterile wax up mix1 (reaction solution added after the sterilized wax is solidified) 3.3 × reaction buffer solution 3.6 μl Enzyme (Tth XL (registered trademark, manufactured by Perkin Elmer)) 0.4 μl up mix2 (sterilized) Reaction liquid to be added after wax is solidified) Template DNA (1.6 ng / μl) 2.0 μl Sterile distilled water 6.0 μl

[0025]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. <Example 1> Synthesis of adapter DNA 1. A DNA having the following sequence was synthesized. (Synthesis by BEX) Sequence 1 5 ′ AGG AAT TCA GCG GCC GCA GAT ATC 3 ′ Sequence 2 5 ′ GAT ATC TGC GGC CGC TGA ATT C 3 ′ Further, these DNAs were purified by a simple cartridge at BEX. I went. The sequence 1 is the sequence shown in SEQ ID NO: 1 in the sequence listing, and the sequence 2 is the sequence shown in SEQ ID NO: 2 in the sequence listing. When annealed, these sequences become double-stranded in the form of formula (1) below. Formula (1) 5 ′ AGG AAT TCA GCG GCC GCA GAT ATC 3 ′ 3 ′ C TTA AGT CGC CGG CGT CTA TAG 5 ′ That is, the 5 ′ end of Sequence 1 is overhanging, and the adapter double-stranded DNA of Formula (1) is It can bind to DNA amplified only at the 3'end of Sequence 1. In effect, this adapter binds to the DNA to be amplified at the phosphorylation site since it phosphorylates the 5'end of sequence 2. The restriction enzyme site of this adapter DNA is shown in FIG.

2. Regarding Sequence 2, the 5'end was phosphorylated under the following conditions. 10 × kinase Buffer A (Nippon Gene) 5 μl Sequence 2 DNA (20 pmol / μl) 25 μl 10 mM ATP 5 μl T4 polynucleotide kinase (10 U / μl) (Nippon Gene) 3 μl Sterile water 12 μl Total 50 μl Reaction at 37 ° C. for 60 minutes Then, denaturation was performed at 75 ° C. for 10 minutes. 3.2. To a solution of the DNA of sequence 2 phosphorylated in step 2, the DNA of sequence 1 prepared at 20 pmol / μl with sterile water was added.
After adding 5 μl and boiling at 95 ° C. for 10 minutes, the mixture was allowed to stand at room temperature for 60 minutes and hybridized to the double strand. This was used as an adapter DNA below.

<Example 2> Amplification reaction of a small amount of DNA by PCR 1. Preparation of DNA to be amplified 1) Cleavage and inactivation procedure of DNA (1) 15 μg of λDNA (Nippon Gene) was cleaved under the following conditions. λDNA 15 μg 10 × H buffer 24 μl (Takara Shuzo) StyI (10 U / μl) 3 μl (Takara Shuzo) Sterile water 198 μl Total 240 μl Cut at 37 ° C. for 8 hours. (2) The inactivation operation was performed at 65 ° C. for 10 minutes. Then, it was adjusted to 50 ng / μl with sterile water.

2). Blunt end The DNA prepared in 1) was blunt ended under the following conditions. Amplified DNA (50 ng / μl) 20 μl 0.5 mM dNTP 10 μl 10 × klenow Buffer 10 μl klenow enzyme (Takara Shuzo) 2 μl Sterile water 58 μl Total 100 μl After 30 minutes of reaction at 65 ° C., denaturation operation was performed. went.

2. Amplified DNA and adapter DNA
Combine 1. The blunted amplified DNA prepared in Example 1 was used.
The adapter DNA prepared in 1.
Ligation was performed using an ion pack. The linking condition is the li
The instructions of the gate pack were followed. Smoothed amplified DNA (10 ng / μl) 20 μl Adapter DNA (6.7 pmol / μl) 4.5 μl BSA (2 mg / ml) (Nippon Gene) 2 μl Hexamine Cobalt Chloride (20 mM) (Nippon Gene) 2 μl Spermidine (20 mM) (Nippon Gene) 2 μl 10 × ligation Buffer (Nippon Gene) 4 μl Sterilized water 3.5 μl Total 38 μl After overnight reaction at 16 ° C., phenol / chloroform extraction was performed, and the recovered aqueous phase was used as a TE solution. The unreacted adapter DNA was removed by applying it to S300 spun clumn (manufactured by Pharmacia) that had been equilibrated with 1. under the conditions according to the instruction manual. Then, the recovered eluate was adjusted to 125 μl with TE solution (1.6 ng /
μl).

3. PCR 2. Using the template DNA to which the adapter was bound by
Amplification reaction of a trace amount of DNA by CR was performed. The temperature during the extension reaction is preferably 68 ° C. at which the reaction proceeds simultaneously with annealing of the primer. 1) Experiment for enzyme selection The enzymes for PCR were Stoffel (registered trademark, manufactured by Perkin Elmer), Ex Taq (registered trademark, manufactured by Takara Shuzo) and Takara Taq (registered trademark, manufactured by Takara Shuzo).
3 types were used. Stoffel is an enzyme suitable for DNA amplification, and Ex Taq is a long-chain DNA amplification enzyme. Takara Taq is an enzyme used in ordinary PCR. The composition of the reaction solution used for PCR is shown in Table 1 (A).
And (B). In the table, the unit of each numerical value is μl except for the tube number. The primer is the DNA of sequence 1.
Where SP is sterile distilled water and B is the reaction buffer. After dispensing each reaction solution, mineral oil was overlaid and PCR was performed. The reaction solutions of tube numbers 1 to 16 were reacted in the following cycle 1, and the reaction solutions of tube numbers 17 to 28 were reacted in the following cycle 2.

[Table 1]

Cycle 1: After reacting at 94 ° C. for 5 minutes, (94 ° C. for 1 minute → 55 ° C. for 1 minute → 72 ° C. for 10 minutes
The reaction was repeated 30 times for 30 minutes. Cycle 2: After reacting at 94 ° C. for 1 minute, the cycle (98 ° C. for 20 seconds → 68 ° C. for 15 minutes) was repeated 30 times for reaction.

After the reaction, 5 μl of the reaction solution was taken from each reaction solution and analyzed by 0.7% agarose gel electrophoresis. This was stained with the fluorescent dye ethidium bromide. The results are shown in FIGS. 2 (A) and 2 (B). The number in each lane shown in the figure corresponds to the tube number of each reaction solution. Also, in each lane, the appearance of each band in each lane is similar to the appearance of the marker band,
This indicates that the DNA amplification is successful. From this figure, it is understood that it is suitable to react under the conditions of tube numbers 17 to 20 or 22 to 27 in order to uniformly amplify short DNA to long DNA. From this, the enzyme used is Stoffel or Ex
Suitability of using Taq, ie DNA
It will be appreciated that amplification enzymes or long chain DNA amplification enzymes are suitable. It can be seen that Ex Taq, that is, a long chain DNA amplification enzyme is particularly suitable. Further, it is found that it is suitable to carry out the reaction in the cycle 2, that is, it is suitable to carry out the extension reaction for 15 minutes at 68 ° C. at which the primer annealing can be carried out simultaneously.

2) Experiment for enzyme selection and uniform amplification condition setting The enzyme for PCR was Stoffel, Ex described above.
Three types of Taq and Tth XL (registered trademark, manufactured by Perkin Elmer) were used. The composition of the reaction solution used for PCR is shown in Tables 2 and 3. In the table, the unit of each numerical value is μl except for the tube number. The primer is DN of sequence 1.
A is A, SP is sterile distilled water, and B is a reaction buffer.
The reaction solutions of tube numbers 29 to 31 were reacted in the following cycle 3, and the reaction solutions of tube numbers 32 to 44 were reacted in the following cycle 4.

[Table 2]

[Table 3]

Cycle 3: After reacting at 94 ° C. for 1 minute, the cycle of (94 ° C. for 20 seconds → 68 ° C. for 15 minutes) was repeated 30 times for reaction. Cycle 4: After reacting at 94 ° C. for 1 minute, the cycle of (94 ° C. for 15 seconds → 68 ° C. for 10 minutes) was repeated 16 times, followed by (94 ° C. for 15 seconds → 68 ° C. for 1 minute).
1 minute) cycle 4 times, followed by (94 ° C. 15 seconds → 68 ° C. 12 minutes) cycle 4 times, followed by (94 ° C. 15 seconds → 6)
The cycle of 8 minutes at 8 ° C.) was repeated 4 times for reaction.

The reaction solutions of tube numbers 35 to 39 were set to hot start, and the reaction solutions of tube numbers 40 to 44 were started by the usual method. Table 3 shows the hot start.
In the reaction liquid of the above, only low mix was dispensed as the reaction liquid, and sterilized wax (the one used was AmpliW
ax (registered trademark, manufactured by Perkin Elmer)), and added.
After 5 minutes at 5 ° C. and then 10 minutes at 4 ° C., up mix1 and up mix2 in Table 3 are added as a reaction solution, and PCR is performed. In actual PCR, the block of the device was preheated to 95 ° C., and then the tube was set to start the reaction.

After the reaction, 6 μl of the reaction solution was taken from each reaction solution and analyzed by 0.7% agarose gel electrophoresis. This was stained with the fluorescent dye ethidium bromide. The results are shown in FIG. The number in each lane shown in the figure corresponds to the tube number of each reaction solution. Further, in each lane, the more similar the appearance of each band in each lane is to the appearance of the band of the marker, the more uniformly DNA amplification is performed, which is preferable.

From this figure, the following can be seen. DNA is uniformly amplified in tube numbers 32 to 3 as compared to tube numbers 29 to 31. That is, cycle 4 is preferable, and it is better that the extension reaction time is gradually increased. Tube number 29 and 32, tube number 3
It should be recognized that the DNA amplification was performed in succession among the groups 0, 31, 33 and 34 and the tube numbers 35 to 44. That is, the enzyme used is St
DNA in the order of offel <ExTaq <Tth XL
Amplification is performed uniformly. That is, long-chain DNA amplification enzyme is more suitable. There should be no difference in DNA amplification between tube numbers 30 and 31 or tube numbers 33 and 34. That is, regarding Ex Taq, no difference was observed depending on the amount of enzyme. Tube numbers 35 and 36 and tube number 3
Between groups 7 to 39 or tube numbers 40 and 4
In both of the groups of 1 and tube numbers 42 to 44, the latter had good DNA amplification. That is, there was a change in DNA amplification depending on the Mg ²⁺ concentration,
Amplification of each DNA should be performed uniformly at a concentration of 1.1 to 1.5 mM. Therefore, for uniform amplification of DNA, it is understood that the condition of using Tth XL to sequentially extend the extension reaction time and adding an appropriate amount of Mg ²⁺ is suitable.

3) Experiment for reaction optimization Tth XL was used as the enzyme for PCR. PCR
Table 4 shows the composition of the reaction liquid used in the above. In the table, the unit of each numerical value is μl except for the tube number. The primer is DNA of sequence 1, SP is sterile distilled water, and B is reaction buffer. The reaction solutions of tube numbers 45 to 48 are reacted in the following cycle 5, and tube number 4
The reaction solutions 9 to 54 were reacted in the following cycle 6. A hot start was set for all samples. In actual PCR, the block of the device was preheated to 95 ° C., and then the tube was set to start the reaction.

[Table 4]

Cycle 5: After reacting at 94 ° C. for 1 minute, the cycle of (94 ° C. for 15 seconds → 68 ° C. for 10 minutes) was repeated 16 times, followed by (94 ° C. for 15 minutes).
Second cycle → 68 ° C. for 11 minutes) cycle 4 times, followed by (94 ° C. 15 seconds → 68 ° C. for 12 minutes) cycle 4 times, followed by (94
The cycle of 15 ° C. for 15 seconds → 68 ° C. for 13 minutes) was repeated 4 times for reaction. Cycle 6: After reacting at 94 ° C. for 1 minute, a cycle of (94 ° C. for 15 seconds → 68 ° C. for 10 minutes) was repeated 16 times, followed by placing at 25 ° C. for 20 minutes and then 9 times.
React at 4 ° C for 1 minute, then (at 94 ° C for 15 seconds → 6
The cycle of 8 minutes at 8 ° C) was repeated 4 times,
Subsequently, a cycle of (94 ° C. for 15 seconds → 68 ° C. for 12 minutes) was repeated 4 times, and then a cycle of (94 ° C. for 15 seconds → 68 ° C. for 13 minutes) was repeated 4 times for reaction.

For the reaction solutions of tube numbers 53 and 54, the cycle of (94 ° C. for 15 seconds → 68 ° C. for 10 minutes) was repeated 16 times, then Ampli w
After the solidification of ax and while standing at 25 ° C., 3.5 μl of a liquid having the following composition was added, and the subsequent reaction was continued. Each component of the added liquid is the same as that added first. Enzyme solution 0.07 μl 3.3 × B 1.0 μl Mg (OAc) ₂ 0.15 μl dNTP 0.28 μl Primer 0.4 μl SP 1.6 μl Total 3 μl

After the reaction, 1 μl and 5 μl were added from each reaction solution.
Each 1 liter reaction solution was taken and analyzed by 0.7% agarose gel electrophoresis. This was stained with the fluorescent dye ethidium bromide. The results of taking 1 μl of the reaction solution are shown in FIG. 1, and the results of taking 5 μl of the reaction solution are shown in FIG. The number in each lane shown in the figure corresponds to the tube number of each reaction solution. Further, in each lane, the more similar the appearance of each band in each lane to the appearance of the band of the marker, the better the DNA amplification is performed.

From these figures, the condition of tube number 45 or 46 is suitable for optimizing the reaction,
It can be seen that the condition of tube number 46 is particularly suitable. From this, the following can be seen. Compared to tube numbers 45 and 46, tube numbers 47 and 48 have well amplified short DNA.
That is, when the primer concentration is high, short DNA is likely to be amplified. The Mg ²⁺ concentration was 1.5 mM rather than 1.1 mM,
Ability to uniformly amplify each DNA. That is, the amount of amplification of each DNA varies depending on the Mg ²⁺ concentration, and 1.5 mM is preferable. There should be no difference between tube numbers 45-48 and tube numbers 49-52. In other words, even if the reaction is stopped for 20 minutes on the way, there should be no apparent difference in the amplification amount of each DNA. Tube numbers 51 and 52 and tube number 5
No difference between groups 3 and 54. That is, even if an enzyme is added in the middle, there should be no obvious difference in the amplification amount of each DNA.

That is, the tube number 4 shown below
If PCR is carried out under the condition of 6, it is possible to simultaneously amplify a wide variety of DNAs from short ones to long ones at the same time. Single-stranded DN of sequence 1 as adapter double-stranded DNA
DNA amplified by hybridizing A and 5'end of sequence 2 with single-stranded DNA phosphorylated
Connect with Single-stranded DNA of Sequence 1 as the primer single-stranded DNA
A is used. Tth XL is used as a DNA amplification enzyme. The PCR reaction was set to hot start, and after reacting at 94 ° C for 1 minute, (94 ° C for 15 seconds → 68 ° C for 1 second
The cycle of 0 minutes) was repeated 16 times, followed by the cycle of (94 ° C. for 15 seconds → 68 ° C. for 11 minutes) 4 times, followed by (94 ° C. for 15 seconds →
A cycle of 68 ° C. for 12 minutes) is repeated 4 times, and then a cycle of (94 ° C. for 15 seconds → 68 ° C. for 13 minutes) is repeated 4 times. The reaction solution having the following composition is used. low mix (first reaction solution) primer (5 pmol / μl) 1.0 μl Mg (OAc) ₂ (25 mM) 1.2 μl dNTP (2.5 mM) 1.6 μl 3.3 × reaction buffer 2.4 μl sterile distillation Water 1.8 μl Sterile wax up mix1 (reaction solution added after sterilization wax solidifies) 3.3 × Reaction buffer 3.6 μl Enzyme (Tth XL) 0.4 μl up mix2 (reaction solution added after sterilization wax solidification) Template DNA (1.6 ng / μl) 2.0 μl Sterile distilled water 6.0 μl

<Example 3> Confirmation of incorporation of primer 1. Preparation of end-labeled primers Primers used for PCR were Megalabels (registered trademark,
Takara Shuzo Co., Ltd.)
A (sequence 1) labeled with end radioactivity was used.
The end radioactivity labeling was performed as follows. Synthetic DNA (Sequence 1) (193 pmol / μl) 3 μl γ-megalabel ³² P ATP (manufactured by Amersham) 5 μl 10 × kination buffer (included in kit) 1 μl T4 polynucleotide kinase (included in kit) 1 μl The reaction solution having the above composition at 37 ° C. After reacting for 30 minutes,
The enzymatic reaction was stopped at 70 ° C. for 10 minutes, and a primer solution was prepared at 5 pmol / μl with sterile distilled water.

2. PCR 1) Using this primer solution, cycle 1 to 4 described above
PCR was performed under the conditions of. Each reaction solution after PCR was diluted 10 times with sterilized water, and 1 μl of each was subjected to 40 V for 2 hours at 0.7 V.
% Agarose gel electrophoresis (using TAE buffer), then 10% TCA (trichloroacetic acid), 10 m
It was soaked in M sodium pyrophosphate solution for 20 minutes, and then dried using a gel dryer model 583 manufactured by Bio-Rad. 2) The dried agarose gel was autoradiographed. Results are shown in FIG. The number of each lane in the figure corresponds to the number of the PCR cycle. From the result, it was revealed that the primer was incorporated almost uniformly up to the portion of about 8 kb in the reaction of cycle 2. This is difficult in the past.
In simultaneous amplification of NA group, regardless of DNA length,
It shows that amplification can be performed almost uniformly.

<Example 4> Amplification of cDNA 1. Synthesis of cDNA 1) Preparation of RNA Total RNA was prepared according to "Methods in enzymol".
"Ogy" Vol. 154 (academic Pres
s Inc. , 1987) pp. 3 to 28. (1) Actually, 3 g of a liver extracted from a rat was ground in liquid nitrogen, and 100 ml of a 5.5 M GTC solution (guanidine thiocyanate 5.5 mM, N-lauronyl sarcosine 0.5%, 25 mM sodium citrate was used. ,
pH 7.0) and homogenized with a potter type homogenizer. (2) The solution was centrifuged at 3000 rpm for 10 minutes, and the supernatant was added to a SW28 swing rotor centrifuge tube (manufactured by Beckman). A cesium trifluoroacetic acid solution (cesium trifluoroacetic acid) having a specific gravity of 1.6 g / ml was added. Acetic acid (Pharmacia) 50%, 100 mM disodium ethylenediaminetetraacetate (EDTA) (pH 7.0)) 1
Layer over 2 ml and use SW28 swing rotor for 2
Separation was performed at 5000 rpm for 24 hours at 15 ° C.

(3) The precipitate was added to 600 μl of 4M GT.
It was dissolved in the C solution and centrifuged at 15000 rpm to collect the solution portion. 15 μl of 1 M acetic acid, ethanol 45
0 μl was added, the mixture was centrifuged at 15,000 rpm for 10 minutes, and the precipitate was collected. (4) This precipitate was added to an appropriate amount (about 3 ml) of TE solution (1
mM Tris-Cl (pH 7.5), 1 mM EDT
Dissolve in A) (TE solution was added until dissolved), 150
The solution portion was recovered by centrifugation at 00 rpm. (5) The same amount of phenol / chloroform as the solution was mixed and centrifuged at 15000 rpm for 10 minutes to collect the solution portion. (6) 1/10 volume of 3M sodium acetate (pH
5.2) was added, and 2.5 times the amount of ethanol was added, and -2
After standing at 0 ° C for 20 minutes, centrifugation was performed at 15000 rpm, the precipitate was washed with 70% ethanol, dried, dissolved in an appropriate amount of TE solution, and stored at -80 ° C. 7 mg of total RNA was obtained from the liver. (7) polyA RNA is oligotex dT
Using 30 super (registered trademark, manufactured by Nippon Roche), the procedure was as instructed. The amount of total RNA used was 1 mg per dose, and oligotex dT30 was used.
PolyA RNA was purified using 750 μl of super. About 20 μg po from 1 mg total RNA
lyA RNA was obtained.

2) Synthesis of cDNA cDNA synthesis is carried out by cDNA synthesis of Pharmacia
The thesis kit was used according to the instruction manual. (1) Using 2 μg of polyA RNA obtained in 1), First strand cDNA and Sec
The ond strand cDNA was synthesized. The enzymatic reaction was performed until the kit was treated with the Klenow fragment and then to the span column purification described in the instruction manual. (2) In the obtained cDNA solution 18.8 μl (6.7 pmol / μl) of the PCR adapter prepared in 1. was added, and the ATP solution included in the kit was added.
n 1 μl and T4 DNA ligase 3 μl were added, and a ligation reaction was performed at 12 ° C. for 16 hours. (3) After incubating at 65 ° C. for 10 minutes to inactivate the enzyme reaction,
To the solution was added 100 μl of Phenol / CHCl ₃ solution and after stirring the aqueous phase was recovered. (4) This solution was added to the STE solution (150 mM NaCl
Was purified by a method described in the kit instruction manual using a span column (equilibrium equilibrated by the method described in the kit instruction manual) equilibrated with a TE solution containing The eluted solution was adjusted to 150 μl with STE solution. This solution was used for the following PCR.

2. PCR of cDNA Table 5 shows the composition of the reaction solution used for PCR. In the table, the unit of each numerical value is μl except for the tube number. The primer is DNA of sequence 1, SP is sterile distilled water, B
Is a reaction buffer. The samples of tube numbers 55 and 56 were used as template DNAs in 1. of Example 3. For the samples of tube numbers 57 to 63 using the DNA prepared in 1., as the template DNA, 1. of Example 4. The cDNA prepared in (the one to which the adapter DNA was bound) was used. With respect to all the samples, the reaction solutions were reacted in the following cycle 7 and used as a hot start. In actual PCR, the block of the device was preheated to 95 ° C., and then the tube was set to start the reaction.

[Table 5]

Cycle 7: After reacting at 94 ° C. for 1 minute, the cycle of (94 ° C. for 15 seconds → 68 ° C. for 10 minutes) was repeated 16 times, followed by (94 ° C. for 15 minutes).
A cycle of second → 68 ° C. for 10 minutes + increase of 15 seconds in each cycle) was repeated 12 times to react, 72 ° C. for 10 minutes, and left at 4 ° C. After completion of the reaction, 1 μl of the reaction solution in each tube was subjected to 0.7% agarose electrophoresis and detection was performed by ethidium bromide staining.

The results are shown in FIG. In the figure, the number of each lane corresponds to the tube number of the sample. As can be seen from the figure, amplification of the DNA prepared in Example 1 showed amplification up to 20 kb, and it was achieved to amplify DNA longer than the DNA confirmed to be amplified by the experimental results so far. It was Further, in the amplification of cDNA, continuous amplification up to about 0.5 to 8 kb was confirmed, and it was confirmed that a cDNA library derived from a living body can be amplified.

[0052]

According to the adapter DNA of the present invention, it becomes possible to bind only one adapter DNA to each end of the amplified DNA in exactly one direction.
Further, it becomes possible to perform PCR accurately without connecting several adapters to each other. In addition, the adapter DNA of the present invention makes it possible to use a commercially available vector for a cDNA library and to easily and efficiently introduce the DNA into any vector. Conventionally, when a wide variety of DNAs are simultaneously amplified, at most 1k
Although only DNA up to about b could be uniformly amplified, the present invention provides an unprecedented effect that even longer DNA can be uniformly amplified. As an example, 0.
A DNA having a length of 5 to 8 kb can be uniformly amplified at the same time. In addition, according to the present invention, a wide variety of
It becomes possible to simultaneously and uniformly amplify a living body-derived cDNA library containing NA.

[0053]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Sequence AGG AAT TCA GCG GCC GCA GAT ATC 24

SEQ ID NO: 2 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Sequence GAT ATC TGC GGC CGC TGA ATT C 22

[Brief description of drawings]

FIG. 1 is a diagram showing the results of analyzing each reaction solution after PCR by 0.7% agarose gel electrophoresis and staining with a fluorescent dye ethidium bromide.

FIG. 2 is a diagram showing the results of analyzing each reaction solution after PCR by 0.7% agarose gel electrophoresis and staining it with a fluorescent dye ethidium bromide.

FIG. 3 is a view showing a result of analyzing each reaction solution after PCR by 0.7% agarose gel electrophoresis and staining with a fluorescent dye ethidium bromide.

FIG. 4 is a diagram showing the results of analyzing each reaction solution after PCR by 0.7% agarose gel electrophoresis and staining with a fluorescent dye ethidium bromide.

FIG. 5: PCR using end labeled primers
It is a figure showing the result of having analyzed each subsequent reaction solution by 0.7% agarose gel electrophoresis, and carrying out autoradiography.

FIG. 6 is a diagram showing the results of performing PCR on rat liver-derived cDNA, analyzing each reaction solution by 0.7% agarose gel electrophoresis, and staining with ethidium bromide fluorescent dye.

FIG. 7 is a view showing restriction enzyme sites of the adapter DNA prepared in Example 2.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 13, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of drawings]

[1] each reaction solution after PCR was analyzed on 0.7% agarose gel electrophoresis, a photograph replacing a drawing surface that represents the results of staining with the fluorescent dye ethidium bromide.

[2] Each reaction solution after PCR was analyzed on 0.7% agarose gel electrophoresis, a photograph replacing a drawing surface that represents the results of staining with the fluorescent dye ethidium bromide.

[3] Each reaction solution after PCR was analyzed on 0.7% agarose gel electrophoresis, a photograph replacing a drawing surface that represents the results of staining with the fluorescent dye ethidium bromide.

FIG. 4 is a photograph instead of a drawing showing the result of each reaction solution after PCR analyzed by 0.7% agarose gel electrophoresis and stained with a fluorescent dye ethidium bromide.

FIG. 5: PCR using end labeled primers
Each reaction solution after was analyzed on 0.7% agarose gel electrophoresis, cash in FIG surface showing the results of autoradiography
It is a bad photograph .

[6] was subjected to PCR for cDNA from rat liver, each reaction was analyzed on 0.7% agarose gel electrophoresis, a photograph replacing a drawing surface that represents the results of staining with the fluorescent dye ethidium bromide.

FIG. 7 is a view showing restriction enzyme sites of the adapter DNA prepared in Example 2.

Claims (12)

[Claims] 1. An adapter double-stranded DN having the following characteristics:
A. One of the single-stranded DNAs forming the adapter double-stranded DNA is projected at one end so that it cannot bind to the amplified DNA or another adapter double-stranded DNA. At the end that binds to the DNA to be amplified, the single-stranded DNA that corresponds to the 5′-end of the single-stranded DNA that constitutes the adapter double-stranded DNA is phosphorylated. 2. A DN that is amplified with a restriction enzyme site that produces a sticky end or a restriction enzyme site that produces a blunt end.
The adapter double-stranded DNA according to claim 1, wherein the adapter double-stranded DNA is contained in a region other than the end not bound to A. 3. The adapter double-stranded DNA according to claim 2, which has at least three restriction enzyme sites that generate sticky ends and restriction enzyme sites that generate blunt ends. 4. The restriction enzyme site for generating sticky ends is E
The adapter double-stranded DNA according to claim 3, which is coRI or NotI, and the restriction enzyme site that produces a blunt end is EcoRV. 5. A hybrid of a single-stranded DNA having the sequence shown in SEQ ID NO: 1 of the sequence listing and a single-stranded DNA having the sequence shown in SEQ ID NO: 2 of the sequence listing and having its 5 ′ phosphorylated at its 5 ′ end. Adapter double-stranded DNA that is soybean. 6. The adapter double-stranded DNA according to any one of claims 1 to 5, which has been chemically modified or enzymatically modified. 7. The amplified DNA among the single-stranded DNAs forming the adapter double-stranded DNA according to claim 1 to 6.
Single-stranded DN whose 5'end is the end not bound to A
A primer single-stranded DNA having a base sequence of A. 8. A single-stranded primer primer having the nucleotide sequence of SEQ ID NO: 1 in the Sequence Listing. 9. A DNA amplification method, which comprises performing a DNA amplification reaction under the following conditions. Ligating with the DNA to be amplified using the double-stranded DNA according to any one of claims 1 to 6 as an adapter DNA. Hybridizing with the template DNA using the single-stranded DNA according to claim 7 or 8 as a primer. Perform an extension reaction using a long-chain DNA amplification enzyme. The PCR cycle is a two-step process. 10. The DNA amplification method according to claim 9, wherein the long-chain DNA amplification enzyme has a high mutation modification ability. 11. A reaction solution having the following composition is first added to low.
add mix, and after the sterilized wax solidifies up mix
The method for amplifying DNA according to claim 9, wherein 1 and up mix2 are added and used. low mix (reaction solution added first) primer (5 pmol / μl) 0.1-1.0 μl Mg (OAc) ₂ (25 mM) 0.88-1.2 μl dNTP (2.5 mM) 1.2-1.6 μl 3.3 × Reaction Buffer 2.4 μl Make up to 8 μl with sterile distilled water. Sterile wax (added to the above) up mix1 (reaction solution added after the sterilized wax has solidified) 3.3 × reaction buffer solution 3.6 μl enzyme (Tth XL (registered trademark, manufactured by Perkin Elmer)) 0.4 μl up mix2 (Reaction solution added after the sterilized wax has solidified) Template DNA 2.0 μl Sterile distilled water 6.0 μl 12. The method for amplifying DNA according to claim 11, wherein the composition of the reaction solution is as follows. low mix (first reaction solution) Primer (5 pmol / μl) 1 μl Mg (OAc) ₂ (25 mM) 1.2 μl dNTP (2.5 mM) 1.6 μl 3.3 × reaction buffer 2.4 μl sterile distilled water 1 Sterile wax up mix1 (reaction solution added after the sterilized wax solidifies) 3.3 × reaction buffer solution 3.6 μl Enzyme (Tth XL (registered trademark, Perkin Elmer Co.)) 0.4 μl up mix2 (sterile wax Reaction solution added after hardening) Template DNA 2.0 μl Sterile distilled water 6.0 μl