Double-stranded vector and process using it

Claim 2

A process for sequencing DNA, which comprises; A. introducing into the insertion site of a double-stranded vector having (a) a first insertion site for foreign DNA that occurs only once in the vector; and (b) a first labelling site that occurs only once in the vector selected from the following types: ______________________________________ (i) --X.sup.3' YX.sup.+ --X.sup.+ Y.sup.+.sub.5 ' X - or --X.sup.3' YZ - --X.sup.+ Y.sup.+.sub.5 ' or Z.sup.+ (ii) --X M Z.sup.3' N.sup.+ Z.sup.+ --X.sup.+ M.sup.+.sub.5 ' Z.sup.+ N X - or --X M Z.sup.3' N.sup.+ Y - --X.sup.+ M.sup.+.sub.5' or Z.sup.+ N Y.sup.+ (iii) --X M.sup.3' N X.sup.+ --X.sup.+ M.sup.+.sub.5' N.sup.+ X - or --M X.sup.3' X.sup.+ N - --M.sup.+ X.sup.+.sub.5' or X N.sup.+ --X M.sup.3' N Y.sup.+ --X.sup.+ M.sup.+.sub.5' N.sup.+ Y - or --M X.sup.3' Y.sup.+ N - --M.sup.+ X.sup.+.sub.5' Y N.sup.+ ______________________________________ in which each of M and N represents a single nucleoside from the group consisting of adenosine, cytidine, guanosine and thymidine, M + and N + represent the nucleosides complementary to M and N, M is not the same as N, each of X, Y and Z is at least one nucleoside selected from the group consisting of adenosine, cytidine, guanosine and thymidine, X + , Y + and Z + represent the nucleosides complementary to X Y and Z, the nucleosides or nucleoside combinations represented by X, Y and Z are not identical to one another and the insertion site (a) and the labelling site (b) are directly adjacent to each other or are separated from each other by up to 1000 base pairs; the DNA to be sequenced; B. cutting the labelling site of the cloned hybrid vector; and C. supplementing the resulting ends of the opened hybrid vector in the presence of DNA polymerase and one or more of the triphosphates dGTP, dTTP, dCTP and dATP, one of the triphosphates being labelled and the hybrid vector is individually labelled; and the labelled and opened hybrid vector is base-specifically degraded and the products of the degradation are analyzed for sequence.

Claim 3

A process according to claim 1, wherein there are used as DNA, overlapping DNA fragments which have been obtained by random degradation of a larger DNA molecule.

Claim 4

A process for sequencing DNA, which comprises: A. providing the DNA to be sequenced, wherein each end of the DNA to be sequenced is further provided with a site that corresponds to one of the two insertion sites of a vector having (a) a first insertion site for foreign DNA that occurs only once in the vector; (b) a first labelling site that occurs only once in the vector selected from the following types: ______________________________________ (i) X.sup.3' YX.sup.+ X.sup.+ Y.sup.+ .sub.5' X - or X.sup.3' YZ - X.sup.+ Y.sup.+ .sub.5' or Z.sup.+ (ii) XMZ.sup.3' N.sup.+ Z.sup.+ X.sup.+ M.sup.+ .sub.5' Z.sup.+ NX - or XMZ.sup.3' N.sup.+ Y - X.sup.+ M.sup.+ .sub.5' or Z.sup.+ NY.sup.+ (iii) XM.sup.3' NX.sup.+ X.sup.+ M.sup.+ .sub.5' N.sup.+ X - or MX.sup.3' X.sup.+ N - M.sup.+ X.sup.+ .sub.5' or XN.sup.+ XM.sup.3' N Y.sup.+ X.sup.+ M.sup.+ .sub.5' N.sup.+ Y - or MX.sup.3' Y.sup.+ N - M.sup.+ X.sup.+ .sub.5' YN.sup.+ ______________________________________ in which each of M and N represents a single nucleoside from the group consisting of adenoside, cytidine, guanosine and thymidine, M + and N + represent the nucleosides complementary to M and N, M is not the same as N, each of X, Y and Z is at least one nucleoside selected from the group consisting of adenosine, cytidine, guanosine and thymidine, X + , Y + and Z + represent the nucleosides complementary to X Y and Z, the nucleosides or nucleoside combinations represented by X, Y and Z are not identical to one another and the insertion site (a) and the labelling site (b) are directly adjacent to each other or are separated from each other by up to 1000 base pairs; and (c) a second insertion site for foreign DNA and, on cutting, one of the first and second insertion sites accepts protruding ends and the other insertion site blunt ends; B. cutting the DNA to be sequenced from a fragment wherein the cut DNA to be sequenced can be exchanged for the DNA fragment resulting when the vector is cut at the insertion sites; C. exchanging the DNA to be sequenced as cut from the provided DNA fragment for the DNA fragment resulting when the vector is cut at the two insertion sites; D. cloning the hybrid vector formed in step C.; E. cutting the labelling site of the cloned hybrid vector; F. supplementing the resulting ends of the opened, hybrid vector in the presence of DNA polymerase and one or more of the triphosphates dGTP, dTTP, dCTP and dATP, one of the triphosphates being labelled, and the hybrid vector is individually labelled; and G. degrading the labelled, opened hybrid vector base-specifically and the products of the degradation are analyzed for sequence.

Claim 5

A process for sequencing DNA, which comprises: A. providing the DNA to be sequenced, wherein each end of the DNA to be sequenced is further provided with a site that corresponds to one of the two insertion sites of a vector having (a) a first insertion site for foreign DNA that occurs only once in the vector; (b) a first labelling site that occurs only once in the vector selected from the following types: ______________________________________ (i) X.sup.3' YX.sup.+ X.sup.+ Y.sup.+ .sub.5' X - or X.sup.3' YZ - X.sup.+ Y.sup.+ .sub.5' or Z.sup.+ (ii) XMZ.sup.3' N.sup.+ Z.sup.+ X.sup.+ M.sup.+ .sub.5' Z.sup.+ NX - or XMZ.sup.3' N.sup.+ Y - X.sup.+ M.sup.+ .sub.5' or Z.sup.+ NY.sup.+ (iii) XM.sup.3' NX.sup.+ X.sup.+ M.sup.+ .sub.5' N.sup.+ X - or MX.sup.3' X.sup.+ N - M.sup.+ X.sup.+ .sub.5' or XN.sup.+ XM.sup.3' N Y.sup.+ X.sup.+ M.sup.+ .sub.5' N.sup.+ Y - or MX.sup.3' Y.sup.+ N - M.sup.+ X.sup.+ .sub.5' YN.sup.+ ______________________________________ in which each of M and N represents a single nucleoside from the group consisting of adenoside, cytidine, guanosine and thymidine, M + and N + represent the nucleosides complementary to M and N, M is not the same as N, each of X, Y and Z is at least one nucleoside selected from the group consisting of adenosine, cytidine, guanosine and thymidine, X + , Y + and Z + represent the nucleosides complementary to X Y and Z, the nucleosides or nucleoside combinations represented by X, Y and Z are not identical to one another and the insertion site (a) and the labelling site (b) are directly adjacent to each other or are separated from each other by up to 1000 base pairs; and (c) a second insertion site for foreign DNA and, on cutting, one of the first and second insertion sites accepts protruding ends and the other insertion site blunt ends; B. cutting the DNA to be sequenced from a fragment wherein the cut DNA to be sequenced can be exchanged for the DNA fragment resulting when the vector is cut at the insertion sites; C. exchanging the DNA to be sequenced as cut from the provided DNA fragment for the DNA fragment resulting when the vector is cut at the two insertion sites; D. cloning the hybrid vector formed in step C.; E. cutting the labelling site of the cloned hybrid vector; F. supplementing the resulting ends of the opened, hybrid vector in the presence of DNA polymerase and one or more of the triphosphates dGTP, dTTP, dCTP and dATP, one of the triphosphates being labelled, and the hybrid vector is individually labelled; and G. degrading the labelled, opened hybrid vector base-specifically and the products of the degradation are analyzed for sequence.

Claim 6

A process according to claim 3, wherein each end of the DNA to be inserted into the vector and to be sequenced is provided with a site that corresponds to that one of the two insertion sites for protruding ends of the vector and the DNA developed in this manner is then degraded to form fragments with one blunt end and where the other end carries one of the original two sites that correspond to the vector insertion site for protruding ends, and the latter end is cleaved.

Claim 7

A process according to claim 3 wherein the DNA to be sequenced has a selection marker.

Claim 8

A process for sequencing DNA, which comprises; A. introducing the DNA to be sequenced into the insertion site of a vector having (a) a first insertion site for foreign DNA that occurs only once in the vector; (b) a first labelling site that occurs only once in the vector selected from the following types: ______________________________________ (i) --X.sup.3' YX.sup.+ --X.sup.+ Y.sup.+.sub.5 ' X - or --X.sup.3' YZ - --X.sup.+ Y.sup.+.sub.5 ' or Z.sup.+ (ii) --X M Z.sup.3' N.sup.+ Z.sup.+ --X.sup.+ M.sup.+.sub.5 ' Z.sup.+ N X - or --X M Z.sup.3' N.sup.+ Y - --X.sup.+ M.sup.+.sub.5' or Z.sup.+ N Y.sup.+ (iii) --X M.sup.3' N X.sup.+ --X.sup.+ M.sup.+.sub.5' N.sup.+ X - or --M X.sup.3' X.sup.+ N - --M.sup.+ X.sup.+.sub.5' or X N.sup.+ --X M.sup.3' N Y.sup.+ --X.sup.+ M.sup.+.sub.5' N.sup.+ Y - or --M X.sup.3+ Y.sup.+ N - --M.sup.+ X.sup.+.sub.5' Y N.sup.+- ______________________________________ in which each of M and N represents a single nucleoside from the group consisting of adenosine, cytidine, guanosine and thymidine, M + and N + represent the nucleosides complementary to M and N, M is not the same as N, each of X, Y and Z is at least one nucleoside selected from the group consisting of adenosine, cytidine, guanosine and thymidine, X + , Y + and Z + represent the nucleosides complementary to X Y and Z, the nucleosides or nucleoside combinations represented by X, Y and Z are not identical to one another and the insertion site (a) and the labelling site (b) are directly adjacent to each other or are separated from each other by up to 1000 base pairs; and (c) a second labelling site of the same type as the first labelling site; B. cloning the hybrid vector formed; C. cutting the two labelling sites of the cloned hybrid vector; D. supplementing the two ends of the cut fragment with the incorporated DNA in the presence of DNA polymerase and one or more of the triphosphates dGTP, dTTP, dCTP and dATP, one of the triphosphates being labelled, whereby only one 3' end of the cut fragment is individually labelled; and E. degrading the labelled cut fragment base-specifically and the products of the degradation are analyzed for sequence.

Claim 9

A process for sequencing DNA, which comprises; A. introducing the DNA to be sequenced into the insertion site of a vector having (a) a first insertion site for foreign DNA that occurs only once in the vector; (b) a first labelling site that occurs only once in the vector selected from the following types: ______________________________________ (i) --X.sup.3' YX.sup.+ --X.sup.+ Y.sup.+.sub.5 ' X - or --X.sup.3' YZ - --X.sup.+ Y.sup.+.sub.5 ' or Z.sup.+ (ii) --X M Z.sup.3' N.sup.+ Z.sup.+ --X.sup.+ M.sup.+.sub.5 ' Z.sup.+ N X - or --X M Z.sup.3' N.sup.+ Y - --X.sup.+ M.sup.+.sub.5' or Z.sup.+ N Y.sup.+ (iii) --X M.sup.3' N X.sup.+ --X.sup.+ M.sup.+.sub.5' N.sup.+ X - or --M X.sup.3' X.sup.+ N - --M.sup.+ X.sup.+.sub.5' or X N.sup.+ --X M.sup.3' N Y.sup.+ --X.sup.+ M.sup.+.sub.5' N.sup.+ Y - or --M X.sup.3+ Y.sup.+ N - --M.sup.+ X.sup.+.sub.5' Y N.sup.+- ______________________________________ in which each of M and N represents a single nucleoside from the group consisting of adenosine, cytidine, guanosine and thymidine, M + and N + represent the nucleosides complementary to M and N, M is not the same as N, each of X, Y and Z is at least one nucleoside selected from the group consisting of adenosine, cytidine, guanosine and thymidine, X + , Y + and Z + represent the nucleosides complementary to X Y and Z, the nucleosides or nucleoside combinations represented by X, Y and Z are not identical to one another and the insertion site (a) and the labelling site (b) are directly adjacent to each other or are separated from each other by up to 1000 base pairs; and (c) a second labelling site of the same type as the first labelling site; B. cloning the hybrid vector formed; C. cutting the two labelling sites of the cloned hybrid vector; D. supplementing the two ends of the cut fragment with the incorporated DNA in the presence of DNA polymerase and one or more of the triphosphates dGTP, dTTP, dCTP and dATP, one of the triphosphates being labelled, whereby only one 3' end of the cut fragment is individually labelled; and E. degrading the labelled cut fragment base-specifically and the products of the degradation are analyzed for sequence.