JPH1066576A - Double-stranded dna having protruding terminal and shuffling method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA having mutations, consisting of a double-stranded DNA having the same sequence as that of a part of a gene and a single-stranded DNA which locates in a discontinuous position with the same or does not locate on the gene and being shuffled by forming a protruding terminal. SOLUTION: This new DNA consists of a double-stranded DNA having the same sequence as that of a part of a gene and a single-stranded DNA having a base arrangement locating at a discontinuous position to the corresponding part of the above- mentioned double-stranded DNA or a base sequence which does not locate on the gene and the single-stranded DNA is connected to one terminal of the double-stranded DNA to form a protruding terminal. By using the new DNA, a DNA containing a different base sequence from a space of a natural base sequence or a mixture of the same is simply obtained, thus, gene products such as excellent proteins, enzymes, etc., can be obtained. This DNA is obtained by preparing the double-stranded DNA by using a part of the DNA as a template and a primer in a DNA polymerase reaction, removing ribonucleotide by enzyme, etc., and further removing nucleotide at 5' terminal side.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a double-stranded DNA having a protruding end comprising a desired sequence and a method for producing the same,
Method for shuffling DNA using DNA block having protruding ends, D shuffled by the method
NA and a DNA pool obtained by application of the method, and a gene product from the pool.

[0002]

2. Description of the Related Art As one approach to protein engineering for improving naturally occurring proteins more usefully for humans, there is an improvement of proteins by site-directed mutagenesis, and some results have been obtained (Japanese Patent Laid-Open Publication No. Hei. No. 91876). However, this requires that the three-dimensional structure of the target protein be known, and much effort is required to analyze the three-dimensional structure. In addition, even if the three-dimensional structure is known, the relationship between the structure and the function is often still unknown, and it is still difficult to reliably provide the desired function.

In order to avoid these difficulties, methods comprising random mutation and screening, and evolutionary molecular engineering utilizing the evolution of living things have been spotlighted and have been shown to be very useful (Proc. Natl. Acad. Sci. US
A, 83 , 576 (1986)). However, the current practice is at most a few amino acid substitutions. WO 95 /
22625 describes a method of randomly dividing a plurality of genes, reconstructing the genes by homologous recombination, and creating a new gene. However, this method is a method for producing a chimeric gene, and the gene to be produced is similar to the original gene, and the outline of the nucleotide sequence is maintained.

[0004] It is difficult for organisms to desire to impart functions that could not be obtained in the course of evolution by such existing methods. In order to obtain gene products whose functions differ greatly from those of gene products such as proteins that exist in nature, a pool of nucleic acids that differ greatly from the naturally occurring nucleotide sequence space is created, and genes with the desired function are selected from those pools. It is considered effective to obtain the product.

As one method for this purpose, it is conceivable to prepare a nucleic acid pool composed of all combinations of bases.
However, even the total number of base sequences (300 bases (bp)) encoding a relatively small protein consisting of 100 amino acids is a huge number of 4 to the 300th power (approximately 10 to the 180th power). It is virtually impossible to prepare a pool.

Attempts have been made to create a mutant in which the order of modules is changed by paying attention to a substructure called a module for a certain kind of protein and changing the order of base sequence blocks corresponding to each module (Viva O.
rigino vol. 23 , No. 1 (1995) 86-87). However, in this attempt, the gene sequences are individually rearranged for each mutant, and a nucleic acid pool containing all the rearranged molecular species is created, and a product of a desired property is obtained from the pool. It has not been possible to obtain a gene to be expressed.

[0007] Using restriction enzymes, several types of DNA blocks having the same protruding end or blunt end are mixed, and a nucleic acid pool composed of various molecules formed by linking them in random order is prepared. It is possible to select a molecule having a desired property from the above. However, such a method requires a restriction enzyme recognition site in DNA, and even if a restriction enzyme recognition site is present, the probability that the site is at a desired position is extremely low. In addition, both ends of the block must have the same cut, and the probability of self-connection is high. Although it may be possible to form a restriction enzyme recognition site by site-directed mutation, whether or not ligation can be performed in frame is largely controlled by chance.
That is, whether or not a protein is synthesized without shifting the reading frame of the genetic code is determined by chance, which is an extremely inefficient method.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently obtaining a base sequence existing in a space greatly different from a naturally occurring base sequence space, and a method for obtaining a naturally obtained base sequence. Is to provide a gene product obtained by expressing a non-existing nucleic acid sequence as a gene.

[0009]

The sequence space of a gene is A,
It consists of all sequences that can be theoretically made using G, C and T bases. For example, a base sequence encoding a protein consisting of n amino acids is constituted by selecting an arbitrary one of four bases 3n times and sequentially arranging the selected bases.
There are n powers. For a protein of 100 amino acids, there are approximately 10 180 corresponding base sequences as described above. In practice, there is no limit on the number of amino acids that make up a protein, so that the sequence space has an infinite extent. In the process of evolution, organisms have only tested a small part of this sequence space, and it is highly probable that there are sequences encoding proteins with excellent functions in other large sequence spaces. The protein engineering efforts currently being carried out by many research institutions are also aimed at creating proteins that are superior in function to naturally occurring proteins, and the main approach is amino acid substitution as described above. However, amino acid substitutions are the primary method used by organisms in evolution,
In other words, it is an imitation of an organism, only searching for the immediate vicinity of the sequence tested by the organism. Also, the sequence obtained in this way may be a sequence that has been selected in the past.

[0010] The present inventors thought that it would be possible to practice the fact that organisms could not do much to get far away from the vicinity of the tested sequence space of the organism. And dividing the gene into several blocks and changing their order,
It was concluded that this method was suitable for this purpose because it was lethal in organisms. As a result of intensive research, they invented a method for creating an arbitrary protruding end on an arbitrary DNA, and by using this method, divided the gene into several blocks and placed them in a sequence space in which their order was changed. The inventors succeeded in creating a molecular pool containing the located nucleotide sequence, and completed the present invention.

That is, the present invention provides the following. 1) (a) a double-stranded DN having the same sequence as a part of the gene
A and (b) the portion corresponding to the double-stranded DNA on the gene comprises a single-stranded DNA having a nucleotide sequence at a discontinuous position or a nucleotide sequence not present on the gene;
A having a protruding end, wherein A is connected to either end of the double-stranded DNA to form a protruding end.
A. 2) (a) a double-stranded DN having the same sequence as a part of the gene
A, (b) a first single-stranded DNA having a nucleotide sequence at a position discontinuous from the portion corresponding to the double-stranded DNA on the gene or a nucleotide sequence not present on the gene, and (c) the gene And a second single-stranded DNA having a base sequence located at a position contiguous with the portion corresponding to the double-stranded DNA above, wherein the second single-stranded DNA corresponds to the continuous position of the double-stranded DNA. A DNA having a protruding end, wherein the first single-stranded DNA is connected to an end of the complementary strand opposite to the end to form a protruding end.

3) The DNA having a protruding end according to 1 or 2, wherein the single-stranded DNA has a length of 2 bases or more. 4) The DNA having a protruding end according to any one of the above 1 to 3, wherein the protruding end is located at the 3 'end. 5) A double-stranded DNA is obtained by performing a DNA polymerase reaction using a part of DNA as a template and an oligonucleotide containing at least one ribonucleotide as a primer.
NA is prepared, and thereafter, ribonucleotides are removed by an enzymatic reaction or a chemical reaction, and nucleotides remaining at the 5 ′ end side of the position where the ribonucleotide was present are further removed. A method for producing a DNA having a terminal.

6) The following steps a) to d): a) (i) an oligonucleotide having a base sequence identical to a part of the gene DNA and (ii) a base sequence of (i) which is discontinuous on the gene An oligonucleotide having a base sequence at a position or a base sequence not present on the gene and containing at least one ribonucleotide such that the oligonucleotide of (ii) is located at the 5 ′ end of the oligonucleotide of (i); B) a step of preparing a double-stranded DNA by performing a DNA polymerase reaction using the DNA containing the oligonucleotide-corresponding portion of a) (i) as a template and the linked oligonucleotide obtained in step a) as a primer; C) the double-stranded DN by an enzymatic reaction or a chemical reaction;
The step of removing the ribonucleotide in A; and the step of d) removing the nucleotide remaining at the 5 'end of the position where the ribonucleotide was present, as defined in 1 above. A method for producing a DNA having a protruding end.

7) The following steps a) to d): a) (i) an oligonucleotide having the same nucleotide sequence as a part of the gene DNA, and (ii) the nucleotide sequence of (i) is discontinuous on the gene. An oligonucleotide having a base sequence at a position or a base sequence not present on the gene and containing at least one ribonucleotide such that the oligonucleotide of (ii) is located at the 5 ′ end of the oligonucleotide of (i); B) using the DNA containing the oligonucleotide-equivalent portion of a) (i) as a template, (i) connecting the oligonucleotide obtained in step a), and (ii) At least 3 'to the 3' end from the oligonucleotide equivalent
A step of preparing a double-stranded DNA by performing a DNA polymerase reaction using an oligonucleotide comprising at least one ribonucleotide, which is a complementary strand of an oligonucleotide located at a position separated by bases or more, as a primer; c) enzymatic reaction or chemical reaction Double-stranded DN
The step of removing ribonucleotides in A; and d) the step of removing nucleotides remaining at the 5 'end of the position where the ribonucleotides existed, respectively. For producing a DNA having protruding protruding ends.

8) DNA having a plurality of DNs having protruding ends
A method of shuffling DNA by dividing into A blocks and connecting these with a sequence different from that before the division. 9) By applying the method defined in any of the above items 5 to 7 to each part of the DNA, the protruding end of one block is complementary to the protruding end of a block that is not adjacent to the original DNA. A method for shuffling DNA by dividing into a plurality of blocks having protruding ends and then ligating with a sequence different from that before the division. 10) The shuffling method according to 8 or 9, wherein the DNA is divided into three or more blocks. 11) The shuffling method according to any one of the above items 8 to 10, wherein the blocks are ligated using DNA ligase. 12) DNA obtained by shuffling according to any one of the above items 8 to 11.

(13) The DNA according to the above (12), which is obtained by shuffling a gene encoding an enzyme function or a control gene thereof. 14) The gene according to 13 above, wherein the gene encodes any one of protease, lipase, cellulase, amylase, catalase, xylanase, oxidase, dehydrogenase, oxygenase, and reductase.
DNA according to 1. 15) The above 13 or 14 wherein the gene is derived from a prokaryote
DNA according to 1. 16) The DNA according to 15 above, wherein the gene is derived from a bacterium belonging to the genus Bacillus. 17) The DNA according to 16 above, wherein the gene is a protease API21 gene.

18) A DNA pool containing a plurality of types of DNAs having different structures obtained by the shuffling method defined in any one of the above items 8 to 11. 19) The DNA pool according to 18 above, which contains 10 or more types of DNA. 20) A mixture of DNA blocks having protruding ends satisfying the following conditions is prepared by applying the method defined in any of the above items 5 to 7 to each part of the template DNA,
A method for producing a DNA pool by linking these with an arbitrary sequence. Condition 1: Each block has a double-stranded portion having the same sequence as a part of the template DNA. Condition 2: Of the blocks that are components of the block mixture,
At least two are complementary to the protruding ends of the blocks that are not adjacent on the template DNA in addition to the double-stranded portion.
It has a main chain portion (protruding end). Condition 3: In the block mixture, the two strands are the same and 1
Includes at least two types of blocks that satisfy condition 2 that differ only in the main chain portion.

21) the template DNA is a gene encoding an enzyme function or its control gene DNA;
3. The method for producing a DNA pool according to item 1. 22) The method for producing a DNA pool according to 21 above, wherein the template DNA is a gene DNA encoding any of protease, lipase, cellulase, amylase, catalase, xylanase, oxidase, dehydrogenase, oxygenase, and reductase. 23) The method for producing a DNA pool according to 22 above, wherein the template DNA is derived from a prokaryote. 24) The method according to the above 2, wherein the template DNA is derived from a bacterium belonging to the genus Bacillus.
4. The method for producing a DNA pool according to item 3. 25) The method for producing a DNA pool according to 24 above, wherein the template DNA is a protease API21 gene. 26) The method for producing a DNA pool according to any one of the above 20 to 25, wherein the DNA blocks are ligated by DNA ligase. 27) A gene product obtained by expressing genetic information of a DNA molecule present in the DNA pool defined in 18 to 26 above.

Hereinafter, the present invention will be described in detail. [DNA having a protruding end] The present invention relates to a DNA having an arbitrary protruding end (hereinafter, unless otherwise specified, "protruding terminal DN").
A ". )I will provide a. Here, the protruding end is 2
A single-stranded portion protruding from the end of the single-stranded DNA. Such a protruding end also occurs when DNA is cut with a restriction enzyme such as EcoRI. In this case, the base sequence of the protruding end is determined by the restriction enzyme, and its length is usually about several bases. When natural DNA is cleaved with a restriction enzyme, the sequence of the double-stranded portion is also limited to the region between the restriction enzyme recognition sites. On the other hand, the protruding end DNA of the present invention has a structure in which a protruding end having a desired length and sequence is added to the end of a double-stranded DNA having an arbitrary sequence.

As described above, the sequence of the double-stranded portion in the terminally protruding DNA of the present invention is not particularly limited. For example, the sequence may be the same as a part of the gene. The length is not particularly limited, but is usually 30 bp or more, preferably 45 bp or more. The sequence of the protruding end is not limited, but is preferably not a sequence having a stem structure in order to prevent self-ligation in various reactions. Here, the “sequence having a stem structure” is a sequence such as AATT whose sequence is exactly the same as its complementary strand (TTAA). The length of the protruding end is usually 2 bases or more, preferably 15 bases or more and 30 bases or less. If the protruding end is too long, a secondary structure is formed and intermolecular annealing becomes difficult. If the length is too short, the melting temperature (Tm) decreases and annealing becomes unstable. The protruding end may be linked to either the 3 ′ end or the 5 ′ end of the double-stranded DNA, but is preferably linked to the 3 ′ end. The structure may have a protruding end only at one end, or may have a structure having protruding ends at both ends.

[Method for Producing Protruding DNA] The protruding DNA of the present invention is typically prepared by the following steps a) to
d). Here, the following equation (1):

Embedded image (Α and c are arbitrary sequences. A and γ are sequences complementary to α and c, respectively.) Based on the double-stranded DNA (template DNA) represented by the following formula (2):

Embedded image (In the formula, β indicates the sequence of the protruding end. The meaning of the other symbols is the same as described above.)
A method for producing A will be described, but terminal-protruding DNA having another structure can be produced in the same manner.

Step a (a-1) Preparation of Oligonucleotide First, a portion to be selected as the double-stranded portion of the protruding end DNA is determined on the template DNA, and the oligonucleotide a complementary to the end α and the other To prepare an oligonucleotide c having the same sequence as the end c. α and c may be sequences having a base length of about 15 to 30.
In addition, an oligonucleotide b complementary to the sequence obtained by removing one base (X) from the 5 'end of the protruding end sequence (β) is prepared. The base sequence β may be a part of the above DNA or an arbitrary sequence not present on the above DNA. These oligonucleotides a, b and c may be obtained by any method. If the sequence is known in advance, it may be synthesized using a known DNA synthesizer.

(A-2) Preparation of Ribonucleotide-Containing Fragment Next, oligonucleotides a and b are ligated via a ribonucleotide. This may be performed by a general synthesis method, but the following method can also be used. First, a phosphate group is added to the 5 ′ end of oligonucleotide a (the following formula:
(3)):

Embedded image (Wherein (P) represents a phosphate group). This reaction is
It can be carried out by the action of polynucleotide kinase. ATP has a molar ratio of 2 to oligonucleotide a.
Use about 10 to 10 times. The reaction temperature is about 30 to 40 ° C, and the reaction time is about 10 minutes to 1 hour. pH 7 ~
About 9 is optimal. The oligonucleotide after the addition of the phosphate group is represented by a '.

Further, a ribonucleotide is added to the 3 ′ end of the oligonucleotide b (the following formula (4)):

Embedded image (In the formula, XTP represents any of ATP, GTP, CTP, and UTP, and (rX) represents a ribonucleotide.) This reaction can be carried out, for example, by the action of terminal deoxynucleotidyl transferase. The nucleoside triphosphate (XTP) to be used includes the step (a-1)
The ribonucleotide corresponding to the base X in is selected. The nucleoside triphosphate is used in a molar ratio of about 2 to 10 times the oligonucleotide b. Reaction temperature is 30 ~
The reaction temperature is about 40 ° C. and the reaction time is about 30 minutes to 2 hours. The oligonucleotide after addition is represented by b '. b '
Is a sequence complementary to the sequence β.

The oligonucleotides a 'and b' thus obtained are mixed, and the 5 'end (phosphate group) of a' is bonded to the 3 'end (hydroxyl group) of ribonucleotide of b' (the following formula: (Five)):

Embedded image This reaction can be performed, for example, by allowing RNA ligase to act in the presence of ATP and a divalent metal ion (Japanese Patent Laid-Open No. 5-292967). Useful divalent metal ions are magnesium ions, manganese ions, etc.
Preferably it is a magnesium ion. RNA ligase can be used as the ligase. RNA ligase is an enzyme that connects RNA with a 3′-terminal hydroxyl group and a 5′-terminal phosphate group. Polydeoxyribonucleotide having only 3′-terminal ribonucleotide and polydeoxyribonucleotide at 5′-phosphate terminal Also connect efficiently. Preferably, T4 RNA ligase is used. The reaction is usually carried out in a buffer at a pH of 7 to 9 and a temperature of 10 to 40 ° C. for 30 to 180 minutes. For example, 50m
M Tris-HCl (pH 8.0), 10 mM MgC
l ₂ , 0.1 mM ATP, 10 mg / l BSA, 1 m
M hexaammine cobalt chloride (HCC), 2
The reaction is carried out at 25 ° C. for 60 minutes or more in a 5% polyethylene glycol 6000 solution.

Step b : Using the DNA containing the sequence α of step (a-1) as a template,
A DNA polymerase reaction is performed using the ligated oligonucleotide b'-a 'obtained in 2) as a primer to obtain a double-stranded D
Prepare NA. Normally, the double-stranded DNA containing the sequences α and γ in step (a-1) on each strand is denatured with heat or alkali to form single-stranded DNA, respectively, and added to b′-a ′ as a primer. PCR is performed using the oligonucleotide c of (a-1). Primer annealing conditions and polymerase reaction conditions are the same as those in general polymerase reactions. Taq polymerase, Klenow fragment (Klenow Fragme)
nt), DNA polymerase I or any other enzyme that catalyzes the DNA extension reaction. As a result of the reaction, the following formula
(6):

Embedded image Thus, a double-stranded DNA having a blunt end represented by

Step c Next, ribonucleotides in the double-stranded DNA are removed by an enzymatic reaction or a chemical reaction. Examples of useful enzymes include ribonuclease. The reaction usually proceeds at pH 6 to 8 and 30 to 70 ° C. for about 10 to 60 minutes. Sodium hydroxide and the like are useful as non-enzymatic drugs. As a result of the reaction, the following equation (7):

Embedded image And a partially discontinuous double-stranded DNA in which the portion corresponding to the base X has been deleted is obtained.

After step d , nucleotides remaining at the 5 'end of the deletion are removed. For example, the double-stranded DNA from which ribonucleotides have been removed in step c is removed by 50%.
It can be carried out by heating to about 90 ° C. Polynucleotides separated from the chain by this operation can be removed using a span column or the like. Thus, a double-stranded DNA with protruding ends represented by the formula (2) can be obtained. In the above description, only one 3 ′ end is a protruding end, but the other 3 ′ end can be a protruding end according to the above method. In the above method, an arbitrary sequence β that is not present at a position contiguous to the sequence α in the template DNA is introduced as a protruding end, but an oligonucleotide present at a position contiguous to the template DNA is used as a protruding end. Is also possible. For example, in the above example, instead of the oligonucleotide c, the oligonucleotide c ′ in which the deoxyribonucleotide at the 3 ′ end is substituted with a ribonucleotide is used as a primer to obtain the following formula (8):

Embedded image May be prepared.

[Method of shuffling DNA]
Further, a DN characterized by using an overhanging DNA
A shuffling method is provided. Here, "shuffling" refers to an operation of dividing DNA and rearranging it arbitrarily. For example, one DNA is represented by the following formula:
(9):

Embedded image _{A-a 1 -a 2 - ......} -a n -B (9) ( may not be leading end A and / or terminal unit B.)
When represented by an array including a connected portion of n blocks as shown in the following expression, shuffling is represented by the following formula:

[Of 10] _{A-a 1 '-a 2'} - ...... -a x -B (10) ( in the _{formula, a 1 ', a 2'} , ......, a x is, a _1, a _2, ...
, A _n are independently selected from the group consisting of an. _{However, a 1 ', a 2'} , ......, the total number of blocks of a _x is a _1, a _2, ......, may not match the total number of blocks of a _n. )).

D using the terminally protruding DNA according to the present invention
FIG. 1 schematically shows the principle of NA shuffling. In FIG. 1, the DNA is not changed for both ends p _A and p _B , and the middle part is changed from p ₁ -p ₂ -p ₃ (top row) to p ₃ -p.
It shuffles to p ₁ -p ₂ (bottom row). Such shuffling is useful as a method for obtaining a gene sequence that has not existed before without changing the sequence of the promoter and the terminator. Specifically, _first , the above-mentioned protruding end DNA method is applied to each part p _A , p ₁ , p ₂ , p ₃ , p _B of the template DNA to form a double-sided protruding type (formula (8)) structure. DNA blocks a ₁ , a ₂ , a ₃ , and DNA blocks A and B having a one-sided protrusion type (formula (2)) structure
Is prepared. The protruding ends a _A and a _1f , a _2f and a _3f are
The complementary strands corresponding to the respective blocks p _A , p ₁ , p ₂ and p ₃ are removed to form protruding ends.

The protrusions a _1r , a _2r , a _3r and a _B are designed according to the desired shuffling arrangement. In the example of FIG. 1, a _1r is designed as a complementary strand of a _3f , and after shuffling, block a ₁ is linked to block a ₃ . Ligation is performed using DNA ligase in the presence of ATP. Regardless of the type of DNA ligase, the single-stranded portion of the protruding end is long, so that it is not necessary to carry out the reaction at usual 16 ° C., and it is advantageous to use a thermophilic DNA ligase. Similarly, in the example of FIG. 1, a _2r , a _3r and a _B are designed as complementary strands of a _1f , a _A and a _2f , respectively. As a result, the structure of the sequence A-a ₃ -a ₁ -a ₂ -B is finally obtained, which apparently divides the DNA before division into blocks p ₁ , p ₂ , p ₃ , p _{3 a,} divided into p _B, it has become _{p a -p 3 -p 1 -p 2} -p same results as those rearranged in the order of _B.

Other arbitrary arrangements can be realized in the same manner as described above. If the blocks A or B are of the both-end projecting type and the other blocks are of the one-end projecting type, shuffling for positioning the other block at the end is also possible. Further, at the time of shuffling, a terminal protruding DNA block not contained in the original gene may be introduced. For example, shuffling can be performed on two or more gene DNAs. In this case, if necessary, the terminal portion of one gene, for example, block A in the above example, may also have a double-ended DNA.
A.

The block which is a unit of shuffling is 2
It is an oligonucleotide or a polynucleotide composed of at least two nucleotides (hereinafter simply referred to as “oligonucleotide”). Usually, it preferably contains 30 or more nucleotide units, more preferably 45 or more nucleotide units. The upper limit of the block length is not particularly limited as long as it is shorter than the length of one gene. However, if the block length is too long, a large portion of the base sequence with no mutation is contained, and therefore, usually 10 to 35% or less of the gene length Is preferred.

When the gene to be shuffled is a gene encoding a protein, it is preferable that the reading frame of the oligonucleotide as the gene block matches before and after the division. In other words, it is desirable that the gene block to be shuffled be designed so that the amino acid sequence generated by translation of the block portion is the same regardless of the relative position after shuffling. For this purpose, a double-stranded portion and a protruding end may be selected in codon units according to the reading frame of the gene DNA. However, it is not necessary to divide into blocks for each semantic segment on the gene. That is, it is not necessary to divide each exon or each segment corresponding to the domain or module of the protein to be encoded. Shuffling at such sites may have been tested in nature in the past. Conventionally, in order to obtain a nucleotide sequence which has not been tested in nature, it is preferable to divide the exon or a site corresponding to a domain or module of a protein to be encoded.

By employing such a technique, it is possible to obtain a protein having an amino acid sequence which has a structure different from that of a natural protein as a whole, but is partially confirmed to be useful in nature. The probability of obtaining a useful protein is higher than that of completely random synthesis.

The type of the gene to be shuffled is not particularly limited. Composed of polynucleotide chains,
Any coding region necessary for expressing a protein or RNA may be used. The nucleotide unit may include any molecule of deoxyribonucleotides or ribonucleotides. For the purpose of finding a useful base sequence, a protein, particularly a gene encoding an enzyme or a gene controlling an enzyme function is preferable. Examples of such enzymes include protease, lipase, cellulase, amylase, catalase, xylanase, oxidase, dehydrogenase, oxygenase, reductase and the like.

The gene may be cloned from a living organism, artificially synthesized, or cloned from a living organism and artificially mutated. Any type can be used as long as it produces a gene product. When it is derived from living organisms, prokaryotes having a clear enzyme-producing ability can be used. Examples of such prokaryotes include Bacillus bacteria, and examples of genes derived from such bacteria include Bacillus NKS-21 (Accession number: FERM).
BP-93-1) (JP-A-5-91876) (SEQ ID NO: 1).

[DNA Pool] The present invention also relates to a DNA pool obtained by applying the shuffling method described above. Here, “DNA pool” refers to two or more types of DNs.
It means a mixture containing A in high density. The DNA pool of the present invention may contain a specific number or more, for example, 10 or more types of DNA molecules having different structures. It is desirable that the reaction can proceed for a plurality of nucleic acid components when used as a mixture in a biochemical operation or reaction, but it does not matter in a state such as a solution state or a dry state. The DNA pool can be produced by preparing a plurality of protruding ends for each block in the shuffling. For example, in the example of FIG. 1, by using not only the complementary strand of a _{3f but also} the complementary strand of other protruding ends a _A and a _2f as the protruding end a _1r of the block a ₁ , A-a ₁ − a ₂ -B, DNA, such as a-a ₁ -a ₂ -a ₁ is obtained. If you add the complement of the other protruding end a _1f of a _1, DNA of A-a ₁ -a ₁ -a ₁ etc., the same block are continuous can also be prepared. Similarly, for the protruding ends of a ₂ and a ₃ , the addition of an oligonucleotide complementary to the other protruding end of the other block or the other protruding end of self enables other sequences including these.

Generally, a certain DNA is referred to as a ₁ , a ₂ , a
_3, divided ..., to the block of a _n. Each block is provided with a protruding end according to the method described above. Overhanging ends are designed as oligonucleotides that are complementary to other blocks or to their own other overhanging ends. Obtained DN
By mixing all or a part of the A blocks and linking them, a nucleic acid pool in which blocks are linked in a random order can be prepared.

[Expression of Genetic Information in Shuffled DNA or DNA Pool] The single-stranded or mixed double-stranded DNA thus shuffled is blunted.
During the ligation reaction, smoothing may be omitted by using a one-end protruding DNA block at the end. For example, the 5 ′ side of a DNA block containing a certain promoter sequence based on the orientation of the promoter is not made a protruding end, but is made a blunt end,
The 3 ′ side of the DNA block containing the terminator sequence based on the direction of the terminator is not set as a protruding end,
Make it blunt. This makes it possible to obtain a gene in which the target gene block is shuffled between the promoter and the terminator, and it is possible to omit the smoothing. Thereafter, the shuffled DNA is incorporated into an arbitrary vector, preferably an expression vector such as pKK223-3, using DNA ligase. In addition, the promoter sequence and the terminator sequence may be one kind or plural kinds.

Alternatively, by forming appropriate restriction enzyme recognition sites in the polynucleotide blocks located at both ends, the restriction enzyme can be used to ligate to a vector. Next, by introducing the vector library prepared as described above into an appropriate host and expressing the genetic information, a gene product having preferable properties and a gene encoding the same can be obtained. The host may be a conventional one. A preferred example is E. coli E.
coli , Bacillus bacteria, yeast, lactic acid bacteria and the like.

However, it is also possible to use an in vitro transcription system and a translation system. In this case, it is possible to express genetic information without connecting to a vector. "Genetic information"
The term “is carried by DNA and refers to that which is translated into a protein or transcribed into RNA in an appropriate organism by itself or by linking to DNA or RNA consisting of another sequence. Although the genetic information expected to be expressed by the method of the present invention is not particularly limited, various gene products, such as enzymes, antibodies, hormones, receptor proteins, ribozymes, and various control functions, such as operators, promoters, antigens, etc. A nuator is an example.

[0043]

The present invention will be described in more detail with reference to the following examples, which do not limit the present invention. Example 1 Production of DNA Pool Protease API21 (Bacillus NKS) shown in SEQ ID NO : 1
-21 (Accession number: FERM BP-93-1)
A nucleic acid pool based on the above-mentioned method was produced by the following procedure. (1) Step a: Preparation of Oligonucleotide Block for Primer (1-1) Synthesis of Oligonucleotide Block Using PerkinElmer's DNA Synthesizer Model 392, 14 kinds of oligonucleotides: oligo FW (SEQ ID NO: 2), Oligo RV (SEQ ID NO: 3), oligo 1r (SEQ ID NO: 4), oligo 1b (SEQ ID NO: 5), oligo 1a
(SEQ ID NO: 6), oligo 2r (SEQ ID NO: 7), oligo 2
b (SEQ ID NO: 8), oligo 2a (SEQ ID NO: 9), oligo 3r (SEQ ID NO: 10), oligo 3b (SEQ ID NO: 11),
Oligo 3a (SEQ ID NO: 12), oligo 4r (SEQ ID NO: 1)
3), oligo 4b (SEQ ID NO: 14), oligo 4a (SEQ ID NO: 15) and oligo A (SEQ ID NO: 16) were synthesized. These are oligonucleotides consisting of or containing a part of the base sequence of API21 (Japanese Patent Laid-Open No. 5-91876) (including a complementary strand). However, oligo 4a is a sequence after glutamine of SEQ ID NO: 1 and includes the termination codon of the gene and the like. These oligonucleotides were used in the subsequent experiments by using Taq polymerase and 3 ′ of the amplified DNA.
It was designed to be optimal when A overhangs at the end. The synthesis of each oligonucleotide was performed with DM trityl-on (ie, the 5 ′ hydroxyl group was protected with a dimethoxytrityl group) and purified with an OPC column.
The reagent used was purchased from PerkinElmer.

(1-2) Addition of ribonucleotide Subsequently, the following composition: 50 mM Tris-HCl buffer (pH 8.0) 10 mM MgCl ₂ 5 mM DTT (dithiothreitol) 25% PEG 6000 1 mM HCC (hexammine cobalt chloride) To a standard solution containing 10 μg / ml BSA (bovine serum albumin), 500 pmol of oligo 1r and A
1 nmol of TP and 10 units of terminal deoxynucleotidyl transferase were added, and the total amount was 1
0 μl was used. This solution was left at 37 ° C. for 1 hour. The same operation was performed for oligo 2r, oligo 3r, oligo 4r, oligo 1b, oligo 2b, oligo 3b, and oligo 4b. The four kinds of polynucleotides generated by this operation were respectively used for oligo 1r ', oligo 2r' and oligo 3
r ', oligo 4r', oligo 1b ', oligo 2b', oligo 3b ', oligo 4b'.

(1-3) Phosphorylation 500 pmol of oligo 1a, 1 nmol of ATP, and 10 units of polynucleotide kinase were dissolved in a standard solution having the same composition as above to make a total volume of 10 μl.
This solution was left at 37 ° C. for 1 hour. The same operation was performed for oligo 2a, oligo 3a and oligo 4a. The polynucleotides generated by this operation are called oligo 1a ', oligo 2a', oligo 3a 'and oligo 4a'.

(1-4) Linking of oligonucleotide blocks 500 pmol of the oligo 1a 'obtained above was combined with oligo 1b', oligo 2b ', oligo 3b' and oligo 4
b ′ 100 pmol each, ATP 1 nmol and
50 units of T4 RNA ligase was added to the above standard solution, and the total volume was made 10 μl, followed by reaction at 25 ° C. for 4 hours.
A similar reaction is performed using oligo 2a ', oligo 1b' and oligo 2
b ', oligo 3b', oligo 4b ', oligo 3a' and oligo 1b ', oligo 2b', oligo 3b ', oligo 4
b ', oligo 4a' and oligo 1b ', oligo 2b', oligo 3b ', oligo 4b'. Oligo 1a ', oligo 1b', oligo 2b ', oligo 3b' and oligo 4b 'produced by these reactions are linked to each other.
Oligo 1M, Oligo 2
a ′ and oligo 1b ′, oligo 2b ′, oligo 3b ′ and oligo 4b ′ were ligated to form a mixture of four kinds of polynucleotides, oligo 2M, oligo 3a ′ and oligo 1b ′, oligo 2
b ', oligo 3b', and oligo 4b 'were ligated together to form a mixture of four kinds of polynucleotides, oligo 3M, oligo 4a' and oligo 1b ', oligo 2b', oligo 3b ', oligo 4
The mixture of the four polynucleotides to which b 'is linked is called oligo 4M.

(2) Steps b to d: Preparation of Gene Block Gene of wild-type alkaline protease cloned from Bacillus NKS-21 is Cla of pHSG396.
Plasmid pSDT812 inserted at the I cleavage site
PCR was performed using (JP-A-1-141596) as a template and oligo 1M and oligo 2r 'as primers. After the gene fragment amplified by this reaction is treated with ribonuclease, it is heat-treated at 80 ° C. for 5 minutes to remove the polynucleotide located on the 5 ′ side from the ribonucleotides present on both strands or one strand. Thereby, a gene block having a protruding end can be prepared. This gene block is called block 1M. The same operation as above was performed using oligo 2M
And oligo 3r ', oligo 3M and oligo 4r', oligo 4
It is also performed with four combinations of M and oligo RV, and oligo FW and oligo 1r '. Block 2M, Block 3 respectively
M, block B, and block F.

Example 2: Equal amounts of shuffling block 1M, block 2M, block 3M, block B and block F were mixed, and a ligation reaction was performed with Pfu DNA ligase. After the reaction, agarose gel electrophoresis was carried out, and the vicinity of 1.5 kilobase pairs was recovered.

Example 3 Confirmation of Nucleic Acid Pool About 1.5 kilobase pairs of the recovered DNA was ligated to the restriction enzyme EcoR.
I, digested with BamHI, and restriction enzymes EcoRI, BamHI
The plasmid was mixed with a plasmid pHY300PLK manufactured by Yakult Honsha which had been digested with HI and treated with alkaline phosphatase, and ligated using a ligation kit (Takara Shuzo). Using this DNA, Escherichia coli JM105 was transformed, and a tetracycline-resistant transformant was selected. Plasmid DNA is extracted, purified, and analyzed from these transformants by a conventional method, and pHY300PLK Eco
1.5 kb DNA between RI and BamHI recognition sites
Thus, 97 clones in which was inserted were obtained. The base sequence of the DNA obtained as described above is analyzed, and block 1 is analyzed.
It was confirmed in what order M, block 2M, block 3M, block F, and block B were connected, that is, how they were shuffled. As theoretically, the block F was located first, the block B was located fifth, and the blocks 1M, 2M, and 3M were shuffled between the two. Table 1 shows the type of each shuffle and the number of clones corresponding thereto.

[0050]

[Table 1] Number of types of shuffle Number of types of shuffle 111 2 223 2 112 5 231 5 113 2 232 2 121 3 233 3 122 4 311 2 123 7 312 6 131 4 313 5 132 5 321 7 133 3 322 2 211 1 323 5 212 5 331 2 213 4 332 5 221 1 333 2 222 3

As described above, when three blocks in a gene are shuffled by the method of the present invention, a nucleic acid pool containing clones of all combinations obtained by selecting three blocks while allowing these blocks to be duplicated can be obtained. It was confirmed that.

Example 4: Gene obtained from nucleic acid pool
Were prepared in selection Example 3 Product DNA were mixed, with this, the Bacillus subtilis (Bacillus subti lis) UOTO999 transformed.
Transformants were selected for tetracycline resistance. When 300 transformants were replicated on a skim milk-containing plate, clear zones could be confirmed around colonies of 12 transformants. This indicates that the enzyme encoded by the shuffled gene can be selected based on the activity. 12 to form these clear zones
As a result of analyzing the nucleotide sequence of the clone, it was found that these were in the same block order as the wild type.

Example 5: Detection of gene product 10 clones selected from the transformants obtained in Example 3 (1 clone is halo-forming, 9 clones are non-forming) and the host Bacillus ( Bacillus) subtilis ) UOTO9
Total RNA was prepared from 99 respectively. In addition, in order to remove the influence of the plasmid in the subsequent hybridization, ribonuclease-free deoxyribonuclease treatment was performed. Subsequently, Northern hybridization was performed using oligo 1r as a probe according to a conventional method. As a result, all bands could be detected in the lane corresponding to the transformant RNA, but no band could be detected in the lane corresponding to the host RNA.

[0054]

According to the present invention, a double-stranded DNA molecule having an arbitrary protruding end can be obtained. In addition, by using this, a DNA containing various base sequences greatly separated from the natural base sequence space or a DNA pool that is a mixture thereof can be obtained by a simple procedure.
It is possible to obtain excellent gene products, such as proteins and enzymes, which cannot be obtained by conventional methods and which have not been tested by organisms in the past. Further, in the method for producing a nucleic acid pool of the present invention, if necessary, a random mixture can be obtained in the middle portion while random shuffling is performed, and a mixture of nucleic acids having a desired sequence fixed to a desired sequence can be obtained. Because the amino acid sequence encoded by can be shuffled without change, compared to a completely random nucleic acid pool production method,
It is likely to produce useful gene products.

[0055]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1122 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Bacillus NKS-21 (Accession number: FERM BP-9)
3-1) Characteristic of sequence Symbol representing the characteristic: sig peptide Location: 1..93 Method for determining the characteristic: S Symbol representing the characteristic: mat peptide Location: 104..1112 Method determining the characteristic: S sequence ATG AAT CTT CAA AAA ATA GCC TCA GCG TTG AAG GTT AAG CAA TCG GCA 48 Met Asn Leu Gln Lys Ile Ala Ser Ala Leu Lys Val Lys Gln Ser Ala -100 -95 -90 TTG GTC AGC AGT TTA ACT ATT TTG TTT CTA ATC ATG CTA GTA GGT ACG 96 Leu Val Ser Ser Leu Thr Ile Leu Phe Leu Ile Met Leu Val Gly Thr -85 -80 -75 ACT AGT GCA AAT GGT GCG AAG CAA GAG TAC TTA ATT GGT TTC AAC TCA 144 Thr Ser Ala Asn Gly Ala Lys Gln Glu Tyr Leu Ile Gly Phe Asn Ser -70 -65 -60 -55 GAC AAG GCA AAA GGA CTT ATC CAA AAT GCA GGT GGA GAA ATT CAT CAT 192 Asp Lys Ala Lys Gly Leu Ile Gln Asn Ala Gly Gly Glu Glu Ile His His -50 -45 -40 GAA TAT ACA GAG TTT CCA GTT ATC TAT GCA GAG CTT CCA GAA GCA GCG 240 Glu Tyr Thr Glu Phe Pro Val Ile Tyr Ala Glu Leu Pro Glu Ala Ala -35 -30 -25 GTA AGT GGA TTG AAA AAT AAT CCT CAT ATT GAT TTT ATT GAG GAA AAC 288 Val Ser Gly Leu Lys Asn Asn Pro His Ile Asp Phe Ile Glu Glu Asn -20 -15 -10 GAA GAA GTT GAA ATT GCA CAG ACT GTT CCT TGG GGA ATC CCT TAT ATT 336 Glu Glu Val Glu Ile Ala Gln Thr Val Pro Trp Gly Ile Pro Tyr Ile -5 1 5 10 TAC TCG GAT GTT GTT CAT CGT CAA GGT TAC TTT GGG AAC GGA GTA AAA 384 Tyr Ser Asp Val Val His Arg Gln Gly Tyr Phe Gly Asn Gly Val Lys 15 20 25 GTA GCA GTA CTT GAT ACA GGA GTG GCT CCT CAT CCT GAT TTA CAT ATT 432 Val Ala Val Leu Asp Thr Gly Val Ala Pro His Pro Asp Leu His Ile 30 35 40 AGA GGA GGA GTA AGC TTT ATC TCT ACA GAA AAC ACT TAT GTG GAT TAT 480 Arg Gly Gly Val Ser Phe Ile Ser Thr Glu Asn Thr Tyr Val Asp Tyr 45 50 55 AAT GGT CAC GGT ACT CAC GTA GCT GGT ACT GTA GCT GCC CTA AAC AAT 528 Asn Gly His Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn 60 65 70 TCA TAT GGC GTA TTG GGA GTG GCT CCT GGA GCT GAA CTA TAT GCT GTT 576 Ser Tyr Gly Val Leu Gly Val Ala Pro Gly Ala Glu Leu Tyr Ala Val 75 80 85 90 AAA GTT CTT GAT CGT AAC GGA AGC GGT TCG CAT GCA TCC ATT GC T CAA 624 Lys Val Leu Asp Arg Asn Gly Ser Gly Ser His Ala Ser Ile Ala Gln 95 100 105 GGA ATT GAA TGG GCG ATG AAT AAT GGG ATG GAT ATT GCC AAC ATG AGT 672 Gly Ile Glu Trp Ala Met Asn Asn Gly Met Asp Ile Ala Asn Met Ser 110 115 120 TTA GGA AGT CCT TCT GGG TCT ACA ACC CTG CAA TTA GCA GCA GAC CGC 720 Leu Gly Ser Pro Ser Gly Ser Thr Thr Leu Gln Leu Ala Ala Asp Arg 125 130 135 GCT AGG AAT GCA GGT GTC TTA TTA ATT GGG GCG GCT GGA AAC TCA GGA 768 Ala Arg Asn Ala Gly Val Leu Leu Ile Gly Ala Ala Gly Asn Ser Gly 140 145 150 CAA CAA GGC GGC TCG AAT AAC ATG GGC TAC CCA GCG CGC TAT GCA TCT 816 Gln Gln Gly Gly Ser Asn Asn Met Gly Tyr Pro Ala Arg Tyr Ala Ser 155 160 165 170 GTC ATG GCT GTT GGA GCG GTG GAC CAA AAT GGA AAT AGA GCG AAC TTT 864 Val Met Ala Val Gly Ala Val Asp Gln Asn Gly Asn Arg Ala Asn Phe 175 180 185 TCA AGC TAT GGA TCA GAA CTT GAG ATT ATG GCG CCT GGT GTC AAT ATT 912 Ser Ser Tyr Gly Ser Glu Leu Glu Ile Met Ala Pro Gly Val Asn Ile 190 195 200 AAC AGT ACG TAT TTA AAT AAC GGA TAT CGC AGT TTA AAT GGT ACG TCA 960 Asn Ser Thr Tyr Leu Asn Asn Gly Tyr Arg Ser Leu Asn Gly Thr Ser 205 210 215 ATG GCA TCT CCA CAT GTT GCT GGG GTA GCT GCA TTA GTT AAA CAA AAA 1008 Met Ala Ser Pro His Val Ala Gly Val Ala Ala Leu Val Lys Gln Lys 220 225 230 CAC CCT CAC TTA ACG GCG GCA CAA ATT CGT AAT CGT ATG AAT CAA ACA 1056 His Pro His Leu Thr Ala Ala Gla Gln Ile Arg Asn Arg Met Asn Gln Thr 235 240 245 250 GCA ATT CCG CTT GGT AAC AGC ACG TAT TAT GGA AAT GGC TTA GTG GAT 1104 Ala Ile Pro Leu Gly Asn Ser Thr Tyr Tyr Gly Asn Gly Leu Val Asp 255 260 265 GCT GAG TAT GCG GCT CAA 1122 Ala Glu Tyr Ala Ala Gln 270 272

SEQ ID NO: 2 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Type of linear sequence: Other nucleic acid Synthetic DNA sequence GATTTTAGAA TTCGCAGCGG

SEQ ID NO: 3 Sequence length: 25 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGGATTCCT TAAAGCCCTG AATAA

SEQ ID NO: 4 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ACAGTCTGTG CAATTTC

SEQ ID NO: 5 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GAAATTGCAC AGACTGT

SEQ ID NO: 6 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCTTGGGGAA TCCCTTATAT

SEQ ID NO: 7 Sequence length: 17 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCCAATACGC CATATGA

SEQ ID NO: 8 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCATATGGCG TATTGGG

SEQ ID NO: 9 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTGGCTCCTG GAGCTGAACT

SEQ ID NO: 10 Sequence length: 16 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCTGATCCAT AGCTTG

SEQ ID NO: 11 Sequence length: 16 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CAAGCTATGG ATCAGA

SEQ ID NO: 12 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTTGAGATTA TGGCGCCTGG

SEQ ID NO: 13 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TGAGCCGCAT ACTCAGC

SEQ ID NO: 14 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCTGAGTATG CGGCTCA

SEQ ID NO: 15 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TAATCCCTAA GGATGTACTG

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing one embodiment of the DNA shuffling method of the present invention.

Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12P 21/02 C12P 21/02 C (C12N 15/09 ZNA C12R 1:07) (C12N 1/21 C12R 1: 125) (C12P 21/02 C12R 1: 125)

Claims (27)

[Claims] (1) a double-stranded DNA having the same sequence as a part of a gene; and (b) a base sequence or said gene located at a discontinuous position on said gene corresponding to said double-stranded DNA. Consisting of a single-stranded DNA having a base sequence not above,
A DNA having a protruding end, wherein the single-stranded DNA is linked to either end of the double-stranded DNA to form a protruding end. 2. (a) a double-stranded DNA having the same sequence as a part of a gene; (b) a base sequence existing at a discontinuous position on the gene from a portion corresponding to the double-stranded DNA or the gene A first single-stranded DNA having a base sequence not present above, and (c) a second single-stranded DN having a base sequence at a position on the gene that is continuous with the portion corresponding to the double-stranded DNA.
A, the second single-stranded DNA has a first single-stranded DN at the end corresponding to the continuous position of the double-stranded DNA.
A is a DNA having a protruding end, wherein the DNA is connected to the end of the complementary strand opposite to the end to form a protruding end. 3. The DNA having a protruding end according to claim 1, wherein the single-stranded DNA has a length of 2 bases or more. 4. The protruding end is located on the 3 ′ end side.
A DNA having a protruding end according to any one of claims 1 to 3. 5. A method using a part of DNA as a template,
A double-stranded DNA is prepared by performing a DNA polymerase reaction using an oligonucleotide containing two ribonucleotides as primers, and then removing the ribonucleotides by an enzymatic reaction or a chemical reaction. A method for producing DNA having a protruding end, comprising removing nucleotides remaining at the 5 ′ end of the DNA. 6. The following steps a) to d): a) (i) an oligonucleotide having a base sequence identical to a part of the gene DNA and (ii) a base sequence of (i) non-contiguous on the gene; An oligonucleotide having a base sequence at a position or a base sequence not present on the gene and containing at least one ribonucleotide such that the oligonucleotide of (ii) is located at the 5 ′ end of the oligonucleotide of (i); B) a step of preparing a double-stranded DNA by performing a DNA polymerase reaction using the DNA containing the oligonucleotide-corresponding portion of a) (i) as a template and the linked oligonucleotide obtained in step a) as a primer; C) the double-stranded DN by an enzymatic reaction or a chemical reaction;
2. The method according to claim 1, further comprising: removing a ribonucleotide in A; and d) removing a nucleotide remaining at the 5 'end of the position where the ribonucleotide was present. For producing a DNA having protruding protruding ends. 7. The following steps a) to d): a) (i) an oligonucleotide having a base sequence identical to a part of the gene DNA and (ii) a base sequence of (i) which is discontinuous on the gene An oligonucleotide having a base sequence at a position or a base sequence not present on the gene and containing at least one ribonucleotide such that the oligonucleotide of (ii) is located at the 5 ′ end of the oligonucleotide of (i); B) using the DNA containing the oligonucleotide-equivalent portion of a) (i) as a template, (i) connecting the oligonucleotide obtained in step a), and (ii) At least 3 'to the 3' end from the oligonucleotide equivalent
A step of preparing a double-stranded DNA by performing a DNA polymerase reaction using an oligonucleotide comprising at least one ribonucleotide, which is a complementary strand of an oligonucleotide located at a position separated by bases or more, as a primer; c) enzymatic reaction or chemical reaction Double-stranded DN
3. The method according to claim 2, further comprising the steps of: removing a ribonucleotide in A; and d) removing each of the nucleotides remaining at the 5 ′ end from the position where the ribonucleotide was present. A method for producing a DNA having a defined protruding end. 8. A plurality of DNAs having protruding DNA ends
A method for shuffling DNA by dividing into blocks and linking them with a different sequence from before the division. 9. The method according to claim 5, wherein each part of the DNA is applied to the DNA by
After dividing the block into a plurality of blocks with protruding ends so that the protruding ends of the block are complementary to the protruding ends of the blocks that are not adjacent to the original DNA, ligate them with a different sequence from before the division. DNA shuffling method according to the above. 10. The shuffling method according to claim 8, wherein the DNA is divided into three or more blocks. 11. The shuffling method according to claim 8, wherein the blocks are linked using DNA ligase. 12. A DNA obtained by shuffling by the method defined in any one of claims 8 to 11. 13. The DNA according to claim 12, which is obtained by shuffling a gene encoding an enzyme function or a control gene thereof. 14. The gene may be a protease, lipase,
Cellulase, amylase, catalase, xylanase,
14. The DNA according to claim 13, which is a gene encoding any of oxidase, dehydrogenase, oxygenase, and reductase. 15. The gene according to claim 1, wherein the gene is derived from a prokaryote.
15. The DNA according to 3 or 14. 16. The DNA according to claim 15, wherein the gene is derived from a Bacillus bacterium. 17. The DNA according to claim 16, wherein the gene is a protease API21 gene. 18. A DNA pool containing a plurality of types of DNA having different structures obtained by the shuffling method defined in any one of claims 8 to 11. 19. The method according to claim 1, which comprises 10 or more types of DNA.
9. The DNA pool according to 8. 20. A mixture of DNA blocks having protruding ends satisfying the following conditions is prepared by applying the method defined in any one of claims 5 to 7 to each part of the template DNA, By connecting with
A pool manufacturing method. Condition 1: Each block has a double-stranded portion having the same sequence as a part of the template DNA. Condition 2: Of the blocks that are components of the block mixture,
At least two are complementary to the protruding ends of the blocks that are not adjacent on the template DNA in addition to the double-stranded portion.
It has a main chain portion (protruding end). Condition 3: In the block mixture, the two strands are the same and 1
Includes at least two types of blocks that satisfy condition 2 that differ only in the main chain portion. 21. The method for producing a DNA pool according to claim 20, wherein the template DNA is a gene encoding an enzyme function or its control gene DNA. 22. The production of the DNA pool according to claim 21, wherein the template DNA is a gene DNA encoding any of protease, lipase, cellulase, amylase, catalase, xylanase, oxidase, dehydrogenase, oxygenase, and reductase. Method. 23. The method for producing a DNA pool according to claim 22, wherein the template DNA is derived from a prokaryote. 24. The method for producing a DNA pool according to claim 23, wherein the template DNA is derived from a bacterium belonging to the genus Bacillus. 25. The template DNA is protease API21.
The method for producing a DNA pool according to claim 24, which is a gene. 26. The method for producing a DNA pool according to claim 20, wherein the DNA blocks are ligated by DNA ligase. 27. The DNA according to claim 18
A gene product obtained by expressing the genetic information of a DNA molecule present in a pool.