JPS5951352A - Two-chain vector and its use method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to double-stranded vectors having a single insertion site for foreign DNA and a single labeling site that can be individually labeled.

Furthermore, the present invention also relates to methods of using the vectors described above.

In fact, the sequence analysis of large molecules I was carried out using the Maxam-Gilbert method and the Sanger-Coulson (8anger-00ulBOn) method.
This has been possible since the development of law. These methods are based on three main principles:

(2) Using labeled nucleotide triphosphates in vitro to label with radioisotopes at or between the 5' or 3' ends of polynucleotides.

(2) Using synthetic or deductive techniques on polynucleotides to arrange them to form groups of four or more fragments with fixed shared ends.

(3) Using denaturing polyacrylamide gel electrophoresis to sort according to the length of the molecules within the fragment groups and copy them.

Therefore, the nucleotide sequence can be revealed in the form of polyacrylamide gel autoradiography results by mutual comparison of the different fractionation patterns of the four groups mentioned above. Each individual analysis can yield sequences of DNA fragments of 200-500 nucleotides. These individual fragments are then classified by testing overlapping ranges or areas.

Large scale sequence analysis relies primarily on the ease of generating fragments and analysis in an appropriate form prior to base-specific reactions. In the Sanger-Cournon method, this problem is solved by the subcloning of random DNA fragments in the vector introduced from bacteriophage M13 at the insertion site in close proximity to the vector-specific sequence, and the sequence reaction is carried out from the insertion site. The solution is in an elegant way: primer-initiated repair synthesis in the presence of dideoxytriphosphate. Single-stranded DNA matrices can be isolated by simple and sophisticated manipulations.

In contrast, the Maxam-Gilbert method requires one or more gel separation steps and fragment-specific enzymatic reactions for introduction and separation of the radiolabel. Therefore, this method often relies on pre-existing sequences of restriction cleavage sites to reduce the number of individual analyses.

Plasmid subcloning systems similar to the M13 system have been developed in various laboratories.

Their purpose is to use chemically degraded sequencing of individual recombinant subclones of the DNA molecules to be sequenced. A basic feature of these sequence vectors is that the two restriction cleavage sites are in close proximity to the subcloning site (insertion site). Sites in close proximity to the insertion site are used to label the reaction, while other sites allow separation of the label. Two labeled fragments are thus formed. The smaller 7 fragments are not removed, resulting in unreadable sequences in the lower regions of the arraying gel. Such loss of sequence information amounts to less than 30 nucleotides in all methods known to date. All these methods still include at least two restriction reactions and at least one labeling reaction.

The object of the invention is to introduce the foreign DNA to be sequenced, or fragments of the foreign DNA to be sequenced, into double-stranded vectors which can be individually labeled in a simple manner. Therefore, it is intended that only one strand of the vector be labeled.

According to the invention, for this purpose, an insertion site (a) for foreign DNA, which is uniquely expressed in the vector described below, is provided.
, and a double-stranded vector having only one labeling site (b) co-extracted in the vector and capable of being individually labeled. This is preferably a labeling moiety of the following type:

-MX"Y"N--M"X+szYN" - where each M and N represents a single base selected from the group consisting of adenosine, cytidine, guanosine and thymidine, and M+ and t represent V and N , M is not the same as N, and each of X, Y and 2
represents one or more bases selected from the group consisting of adenosine, cytidine, guanosine and thymidine, and ! +,! + and 2+ represent bases that complement X, Y and 2, and X,! The bases or combinations of bases represented by and 2 are not identical to each other and are of type (i
), Y is not the same as Y .

Double-stranded vectors are circular pieces of DNA that can be cloned.

Preferably, the insertion site (a) and the labeling site (1)) are directly adjacent to each other or are separated from each other by up to 1000, preferably 100 and especially 10 base pairs. The insertion site (a) may be one for the introduction of foreign DNA with a protruding or blunt end.

The method for sequencing foreign DNA is as follows.

- Introducing the foreign DNA to be sequenced into the insertion site of the vector; - Cloning the hybrid vector formed; - Cleaving the marker site (b) of the cloned hybrid vector; - Generation of an open hybrid vector. The ends are treated with DNA polymerase and one or more triphosphates (I
GTP, dTTP, dOTP and dATP are supplemented in the presence of labeled triphosphate (e.g. with a radioactive isotope), thereby individually labeling the hybrid vector; The base-specific degradation is performed in situ using a method, and the degradation products are analyzed in situ using known methods.

Of course, overlapping DNA fragments obtained by random decay of relatively large NA molecules can be used as foreign DINA according to the method of the invention.

In the vector according to the invention, the insertion site (,) has a different function from the labeling site (b). The insertion site (a) is used to receive the foreign DNA to be sequenced, while the labeling site (b) is used for individual labeling of the open hybrid vector.

The individual labeling of hybrid vectors according to the invention is explained diagrammatically and in detail (by way of the following example).

Labeled site dGTη (IOTP, supplemented with dATP -T
TTOAAA --AAAGTT
T -dGTP, supplemented with dTTηdOTP -TT
TOTTOAAA--AAAGTT GTTT
- cost tlQTP, dTTη (supplemented with io'f'P -TT
TOTTOAAA--AAAGTT GT'l
"T-dG'l'P;aT'I'P; Supplement with dATP -TTT TTAAA ---muAAGTT
AAGTTT −dTTηdOTηtlAT
Supplement with P -TT'l'OAA TTAAA-
-AAAGTT TTT- Note: G = Nibuanosine T = Thymidine, C = Cytidine, A
= Adenosine, Stain = Label From the selected examples, one skilled in the art would label triphosphates, or label combinations of triphosphates.
It can be seen that, based on the properties of the known labeling sites with which the triphosphates are labeled within the combination, it can be foreseen that individual labeling at a given labeling site can be achieved. In the examples given, this is the case for triphosphate mixtures dGTP, dTTP and aa"TP or dG"-rich dTTP and dOTP.

According to another sequence of the invention, the double-stranded vector has the following sites:

- Insertion site (a) for foreign Dl that is uniquely found in the vector; - Can be individually labeled and is uniquely found in the vector.
The label site (b) to be found, for example, the above-mentioned type (i
), (ii) or C1D labeling site (b): - Additional insertion site for foreign DNA found only once in the vector (, ).

Then, at the same time as cutting, two insertion sites (a) and (
'), one results in a protruding end and the other results in a blunt end.

Preferably, the two insertion sites (a) and (0) are located on the same side as the labeling site (b) and are in close proximity. Insertion sites (a) and (0) are preferably directly adjacent to each other or separated from each other by up to 1000, preferably 100, especially 10 base pairs.

An example of such a vector is pO8Vo5, which is described in detail below.

In such an arrangement of the present invention, the method for sequencing foreign DNA is as follows.

- 2 of the vector at each end of the foreign DIJA to be sequenced
provide an insertion site corresponding to one of the two insertion sites (a) and (0); - destroy the foreign DNA generated in such a way,
Forming a fragment that can be exchanged with the DNA fragment generated when insertion sites (a) and (0) of the vector are cleaved; Cutting foreign DNA from between the two insertion sites (a) and (0) of the vector - Clone the formed hybrid vector; - Cut the labeled site (1)) of the cloned hybrid vector; - Cut the generated end of the opened hybrid vector with DNA polymerase and one Or even more Triphos7 x −+
LGTP, dTTP, dOTP and (LA
TP (where at least one triphosphate is e.g. by radioactivity) in the presence of triphosphates and individually labeling the hybrid vectors; The base-specific degradation is performed in situ using a method, and the degradation products are analyzed in situ using known methods.

Vector insertion site (a) -! with an insertion site corresponding to bamboo (C) (preferably for a protruding end);
The features that give each end of the foreign DNA to be sequenced can be similarly expressed as follows. That is, the two ends of the foreign DNA to be sequenced are provided with linkers.

According to another sequence of the invention, the double-stranded vector has the following sites:

- an insertion site (a) for the foreign DNA which is found only once in the vector; - a labeling site (b) which is found only once in the vector and can be individually labeled, for example type (1) as described above.

Label site (b) of (11) or (iii); - An additional label site of the same type as the first label site (b), found only once in the vector.

Preferably, the two labeling sites (1)) and (d) are located on the sides of the labeling site (a) and are arranged in close proximity thereto. Preferably, two labeling sites (b) and (d
) are directly adjacent to the insertion site (a) or are separated from it by up to 1000, preferably 100 and especially 10 base pairs.

As already explained, two labeling sites (1)) and (
d) should be found only once in the vector.

However, it would be advantageous if they could be cleaved by the same restriction enzyme. The insertion site (,) may be one for insertion of foreign DNA with a protruding or blunt end.

A method for sequencing foreign DNA having a vector with such a sequence is as follows.

・Insert the foreign DNA to be sequenced into the vector insertion site <)
- Clone the formed hybrid vector: - Two labeling sites (b) and (
d) cutting; 2 of the cut fragments with the foreign DNA introduced;
DNA polymerase and one or more triphosphates) dGTP.

dTTP, dOTP and aArp (at least 1
The labeled cleavage fragments are base-specific in situ using known methods, while individually labeling one end and the other end of the cleavage fragment. and the degradation products are analyzed in situ using known methods.

The individual labeling according to the invention of cleavage fragments with introduced foreign DNA is explained in detail diagrammatically by the following example.

GOTO--OA(j(jT 0GOTG --0AGOT From the selected examples, one skilled in the art would have labeled the triphosphate, or labeled a combination of triphosphates, and labeled the triphosphate within said combination.
Based on the properties of known labeling moieties (b) and (d),
It can be seen that it is foreseeable that individual labeling at one of the two labeling sites can be achieved. This example shows that either one or the other of the labeling sites can be labeled individually. Thus, in such an arrangement of the invention, both strands of foreign DNA can be sequenced. The two strands can be perfectly aligned, thus increasing the reliability of sequence analysis. However, it is equally possible to arrange the two strands within the regions of overlap common to the two strands. Such methods are particularly advantageous when sequencing very long foreign DNA strands.

In order to create a vector according to the invention, a person skilled in the art can use the DN
It must be possible to arrange, cleave, and combine A molecules. Sequencing methods available to the person skilled in the art have already been described at the beginning. For cleaving and joining DNA molecules, see DE 2814039.8 and the references cited therein.

Both the insertion sites (a) and (e) and the labeling sites (b) and (d) of the vector according to the invention can be cleaved by restriction endonucleases, ie restriction enzymes. Those skilled in the art are also familiar with the random disintegration of large NA molecules into smaller fragments, which can be carried out physically (e.g. using ultrasound) or chemically (e.g. enzymatically). can.

Thus, in accordance with the present invention, for a single restriction reaction and a completed (terminated) labeling reaction, there are many Novel sequence vectors are provided. According to this method,
No duplicate unreadable areas are formed. Furthermore, the subcloning method according to the invention does not result in virtually any tangible or covert clones. A suitable prototype for sequencing plasmids is described in detail below, p08V.
It is called O3.

Construction of p08VO3 pGVOOl is a 1882 bp derivative of pBR327. This converts pBR527 into Hlnd[1 and Ava
Cut at I to complete (terminate) the protruding end;
and formed by automatically joining the blunt ends. pGVOOl carries the origin of replication and the β-lactamase gene. individual IIfcpRI,
The Ota I and Hlnd [1 sites are located 3 posterior to these regions. p08VO3 is a chemically synthesized DIJA fragment (10'bpda)
The completed (terminated) Ol was formed by introducing Ota into the site.

tal p08VO3 is FicoRI, Sma I, Eca
l (=Batl!fll) and IF! 1ncl m
Contains individual cleavage sites for.

Cleavage with lea I (=Bst E II) generates a linear vector molecule with two different overhanging 5'-ends.

10OOGG” “pGTOAOo---4
G G C! OOA G T Opg /
3tG□dGTP, dTTP and alpha”
A completed reaction in the presence of 2P-40'f'P labels only the left end.

The right end cannot be labeled because dATP has been dropped from the reaction mixture.

The subcloned sequence pO8VO3 exhibits the following characteristics.

(1) Kcal or Bst B II cleavage sites, which can be individually labeled by a single completion reaction.

(2) 8mal cleavage site, which overlaps with the Baa 1 site; Sma I can be used as an insertion site with a blunt end. Each fragment introduced into this site is adjacent to Fica I, so it can be individually and easily labeled at this site.

(3) KcoR1 site 23bp0 far from the Sma1 site and facing Baa I. This vector is available from Inc.

It is cut twice with R1 and sma I and can be used to accommodate subcloning of size-fractionated DNA fragments and allows continuous sequencing. refer to the following.

(4) treated with phosphatase and sma I
A small (1894 bp) linearized vector can be preliminarily and easily purified on a low melting point agarose gel (2-inch). Vectors purified in this manner yield hidden clones below 1 as a result of self-ligation.

(5) Selection for transformation depends on the gene for ampicillin resistance.

Subcloning in pO8VO3 Random subcloning (shotgun subcloning): fragments with blunt ends from restriction reactions, random restriction products with deoxyribonuclease (i.e. random deoxyribonuclease digests),
or DNA exposed to ultrasound wavelength: dephosphorylated vector cut with Sma I: binding (l
transformation) reaction; transformation.

Adapted subcloning: combination of Eco R,l linkers with DMA molecules to be aligned; exposure of DNA to ultrasound wavelengths or treatment with deoxyribonuclease; cleavage with lco Rl; on low melting point agarose gel (2T) Fractionation with: Eco R
I - Combination of size fractions using p08VO3 treated with Sma I; Transformation of individual fractions.

Only the fragment with the first terminus can be ligated to the vector. This is because they solely carry the adhesive Kco RI terminus on one side. The different gel fractions therefore form a group of overlapping sequences.

Initial DNA fragments: Thick arrows represent alternating fragments. Note: A DNA molecule containing one or more KcoR1 sites is called Boo.
It can be used without adding an Rl linker. This molecule is made circular before being exposed to ultrasound wavelengths. Bco RI cutting must be performed subsequent to exposure to ultrasound wavelengths.

Sequence of pO8Vo 30 Recombinant Subclone 1. Pick colonies, 1-Grow culture overnight.

2. Centrifuge and lyse the cells (e.g. Quigley and Holmes
) and then clarified to remove impurities.

5. Obtain recombinant DNA.

4. Cleavage is performed using Bst El n (commercially available enzyme).

5 The completion reaction is carried out using Frenara polymerase and dGTP, dTTP and alpha'P-(1(!TP).

& Sephadex (5ephadex) G 50
Perform rapid centrifugation in (1yd).

Z Starts a chemical degradation reaction.

Production of p08v31 p08V31 is produced by cleaving pO8VO3 with restriction enzyme Sma I. A chemically synthesized double-stranded DNA fragment (20 bp) with the following sequence:
) into the cut site.

TGAOTAAGTOGAOTOAGTOAM0TGA
TTOAGOTGAGTOAGT two labeling sites (b)
and (d) differ by two different overhanging ends. In summary, pO8V31 is characterized by the following points:

(1) 8al l cleavage site as initial insertion site (a).

(2) Tthm I cleavage site as labeling site (b).

(3) Tthll1 cleavage site as labeling site (d).

(4) (b) and (d) differ with respect to their arrangement.

(5) F as insertion site (C)! coR1 cleavage site.

(6) Ampicillin resistance gene.

(7) A length of 2000 base pairs or less.

The meanings of the abbreviations used in this specification are as follows.

pBR527: 4 1983 as DSM 2629
It was deposited on the 22nd of May. genes, 9 (198
b) 287-305 bp = base pair d day = double-stranded dGTP: deoxyguanosine triphosphate dTTP: deoxytemidine triphosphate d
OTP: Deoxycytedine triphosphate dA
TP: Deoxyadenosine triphosphate Also, throughout this specification, the term "base" for A (adenosine)%0 (cytidine), G (guanosine) and T (thymidine) is to be understood as "nucleoside."

Agent Patent attorney (8107) Kiyoshi Sasaki (and 3 others) Continued from page 1 Priority claim @September 10, 1982 ■West Germany (DE
) [Yes] P3233689.6 Procedural amendment (method) % formula % ) 1. Indication of the case 1982 Patent Application No. 0130272 2. Name of the invention Double-stranded vector and method of using the same 3. Make amendments Relationship with the case: Patent applicant: Month, Day, Showa (shipment date; Month, Day, Showa) 6, Number of inventions increased by amendment: 0

Claims (1)

[Scope of Claims] 1) (a) an insertion site for a foreign DIm that is uniquely found in the vector; and (b) a label site that is uniquely found in the vector and that can be individually labeled. A double-stranded vector with 2) The following form: (i) -X" YX+- or -Xten Y+sl X - -X" YZ - + x +y +,, z +- or (fi
) −XM zv , +x+−−X+M+
, I Z"MX- or -XM Z"'
N+Y --X"M+, I Z"NY
10- or OiD -xyt3'N x + - or 4+M9I N+X- -M
5. Go Mata 1-XM”
NY+- -X"M", / N1Y-Mataken-MX"
Y1N- -M+X+sZ YN+- (where each M and N represents a single base selected from the group consisting of adenosine, cytidine, guanosine and thymidine, and M+ and N+ represent the bases complementary to M and N) !+
,Y+ and 2+keX,! and represents a base complementary to 2, the bases or combinations of bases represented by X, Y and 2 are not identical to each other, and in the case of type (i) Y is not identical to Y. ) The vector described in 1) above, which has a labeling site.
□ 3) characterized in that the insertion site (,) and the labeling site (b) are directly adjacent to each other) or are separated from each other by up to 1000, preferably 100, in particular up to 10 base pairs. The vector described in the preceding paragraph 1) or 2). 4) The insertion site (a) according to any one of 1) to 3) above, wherein the insertion site (a) is for insertion of foreign DNA having a protruding end or a blunt end. vector. 5) has an additional insertion site (C) for foreign DNA;
Any of the preceding items 1) to 4), characterized in that upon cutting, one of the two insertion sites (a) and (Q) provides a protruding end and the other provides a blunt end. The double-stranded vector according to item 1. 6) The two insertion sites (, ) and (0) are labeled site (b
The vector according to item 5), wherein the insertion site which is present on the same side as and in close proximity to the labeling site and which produces a blunt end upon cleavage is preferably located near the labeling site. 7) The two insertion sites (a) and (c) are directly adjacent to each other or 1000, preferably 1
The vector according to item 5) or 6) above, characterized in that the vectors are separated by up to 00 base pairs, particularly up to 10 base pairs. 8) The following features: SmaI cleavage site as initial insertion site (a), E! as labeling site (b). ca I or Bstll [cleavage site, Eico Rl cleavage site as insertion site (0), ampicillin resistance gene, and any of the preceding items 5) to 7) characterized by having a length of 2000 or less base pairs. The vector according to any one of the items. 9) Any one of the preceding items 1) to 4), characterized in that it has an additional labeling site (d) that is uniquely found in the vector and is of the same type as the first labeling site (b). The double-stranded vector described in. 10) The two labeling sites (b) and (d) are located at the insertion site (
The vector according to item 9) above, which is located on the side of a) and arranged in close proximity thereto. 11) The two labeling sites (b) and (d) are located at the insertion site (
9) or 10), characterized in that it is directly adjacent to a) or separated from it by up to 1000, preferably 100, especially 10 base pairs.
Vectors listed. 12) The following features: 5atI cleavage site as initial insertion site (a), Tth 1111 cleavage site as labeling site (b), Tth m l cleavage site as labeling site (d), (1)) and ( d) differs with respect to their sequences, has a uco Rl cleavage site as an insertion site (0), an ampicillin resistance gene, and a length of 2000 base pairs or less in the preceding items 9) to 11) The vector according to any one of . 13) Next step: (i) Arrange the foreign DNA to be sequenced in the previous sections 1) to 4).
Insertion site (a) of the vector according to any one of
(ii) cloning the formed hybrid vector; cleaving the GiO labeled site (■) of the cloned hybrid vector; or even more triphosphates dGTP, dTTP, dOTP and (IATP)
(wherein at least one triphosphate is eg radioactively labeled) and individually labeling the hybrid vector; 1. A method for sequencing foreign DNA, which comprises base-specific degradation on the spot using the method described above, and analyzing the degraded product on the spot using a known method. 14) The method according to item 13), characterized in that overlapping DNA fragments obtained by random decay of relatively large I) HA molecules are used as the foreign DMA. 15) Next step: (i) Add 5 above to each end of the foreign DNA to be sequenced.
(11) Providing an insertion site corresponding to one of the two insertion sites (a) or (0) of the vector described in any one of ) to 8); (iii) disintegrating the foreign DNA to form a fragment that can be exchanged for the I) NA fragment produced when the insertion sites (a) and (C) of the vector are cleaved; replacing the DNA fragment cut from between a) and (C); Gv) the gene for cloning the formed hybrid vector; and (V) the labeling site (1) of the cloned hybrid vector.
)), generating an open hybrid vector (Vll).
supplemented in the presence of l'P (wherein at least one triphosphate is labeled, e.g. radioactively);
and individually labeling the hybrid vector, and ~1) base-specifically degrade the labeled and opened hybrid vector in situ by a known method, and degrade the degradation product in situ by a known method. 1. A method for sequencing foreign DNA, which comprises analyzing foreign DNA by: 16) Next step: (i) Add 5 above to each end of the foreign DNA to be sequenced.
) to 8), (ii) providing an insertion site corresponding to one of the two insertion sites (1) or (C) for the overhanging end of the vector according to any one of (ii) such Item 15 above, characterized in that the foreign DNA generated by the method is degraded to form a fragment having a blunt end and an end carrying the insertion site, and the insertion site is cut (before or after disruption). ) method described. 17) The method according to item 15) or 16), which uses a foreign DMA having a selective genetic marker (preferably close to the insertion site of the foreign DNA). 1B) Next step: (i) Arranging the foreign DNA to be sequenced in the previous section ~ 11)
(ii) cloning the formed hybrid vector; 611) two labeling sites of the cloned hybrid vector); and (d) cutting the two ends of the cleavage fragment with introduced foreign DMA into D
NA polymerase and one or more triphosphates) dGTP. (ITTP, dOTP and aArp (wherein at least one triphosphate is e.g. radioactively labeled) and only one end of the cleavage fragment on the one hand and the other Individual labeling of only one end, base-specific degradation of the 0-labeled cleavage fragments in situ using known methods, and analysis of the degradation products in situ using known methods. A method for sequencing characteristic foreign DNA.