EP0040466A2
Expired / Lapsed
EP
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Adaptor molecules for DNA and their application to synthesis of gene-derived products
Assignee: National Research Council Of Canada Te Ottawa Can
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Filing: 1981-03-26
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Granted: —
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Expires: 2001-03-26
Abstract
Abstract Adaptor molecules have been prepared to comprise either start or stop signals for protein synthesis, in addition to recognition sites for restriction endonucleases. Separate adaptors may be used in a symmetrical duplex form. The start adaptor may include nucleotide base inserts to provide the correct reading frame of the triplet code in a DNA sequence with inappropriate reading frame. Insulin A-chain and B-chain genes of the human type, have been synthesized with the appropriate adaptor molecules provided on each end. The adapted DNA genes have been joined to replicable cloning vehicles and the hybrid DNA transferred to a host cell. The transformed host cell has been shown to contain the desired insulin gene.
Inventors
- 👤 Saran A. Narang
- 👤 Ray J. Wu
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 17:57
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/62; C12N15/63; C12N15/66; C12N15/70
Claims Analysis
Total Claims: 82
Claim 1
Independent Claim
72 words
Adaptor oligodeoxynucleotide molecules, for facilitating insertion of DNA information sequences into a cloning vehicle, and transcription and translation of said sequences in a host cell; each adaptor comprising: (a) a recognition site for a restriction endonuclease, and (b) a signal for protein synthesis, selected from a start signal and a stop signal, said start signal being downstream of said recognition site and said stop signal · being upstream of said recognition site.
Claim 2
Dependent Claim
72 words
Adaptor oligodeoxynucleotide molecules, for facilitating insertion of DNA information sequences into a cloning vehicle, and transcription and translation of said sequences in a host cell; each adaptor comprising: (a) a recognition site for a restriction endonuclease, and (b) a signal for protein synthesis, selected from a start signal and a stop signal, said start signal being downstream of said recognition site and said stop signal · being upstream of said recognition site.
Claim 3
Dependent Claim
36 words
The start signal adaptor molecules of claim 1 including at least one additional base between each start signal and the recognition site to correct the frame shift of a DNA information sequence with inappropriate reading frame.
Claim 4
Dependent Claim
42 words
Adaptor molecules of claim 1 which are symmetrical DNA duplexes having duplicate signals at each end, each complementary nucleotide strand having complete palindromic symmetry to its opposite strand, with both halves of the duplex being identical on 180° rotation of one half.
Claim 5
Dependent Claim
34 words
Start signal adaptor molecules of claim 3 including additional nucleotide bases between each start signal and the central recognition site, to correct the frame shift of a DNA information sequence with inappropriate reading frame.
Claim 6
Dependent Claim
16 words
The adaptor molecules of claim 3 wherein from 8 to 20 nucleotide base pairs are present.
Claim 7
Dependent Claim
20 words
The start signal adaptor molecule of claim 1 wherein said start signal comprises the ATG codon or the GTG codon.
Claim 8
Dependent Claim
27 words
7 . The stop signal adaptor molecules of claim 1 wherein said stop signal comprises at least one codon selected from the group TGA, TAA, and TAG.
Claim 9
Dependent Claim
13 words
ε. The adaptor molecule of claim 3 comprising one of the duplex structures:
Claim 12
Dependent Claim
10 words
The start signal adaptor of claim 8 comprising the structure:
Claim 13
Dependent Claim
10 words
The start signal adaptor of claim 4 comprising the structure:
Claim 14
Dependent Claim
10 words
The start signal adaptor of claim 4 comprising the structure:
Claim 15
Dependent Claim
25 words
The start signal adaptor of claim 6 including a strong promoter sequence for mRNA transcription located upstream from the start signal and the recognition site.
Claim 16
Dependent Claim
13 words
The stop signal adaptor of claim 7 comprising one of the duplex structures:
Claim 17
Dependent Claim
15 words
The stop signal adaptor of claim 13 wherein the recognition site has the following structure:
Claim 18
Dependent Claim
11 words
The stop signal adaptor molecule of claim 7 having the structure:
Claim 19
Dependent Claim
11 words
The stop signal adaptor molecule of claim 7 having the structure:
Claim 20
Dependent Claim
17 words
An adaptor molecule of claim 1 ligated to a DNA information sequence to give an adapted DNA.
Claim 21
Dependent Claim
18 words
The adapted DNA information sequence of claim 17 wherein said sequence is a gene or a partial gene.
Claim 22
Dependent Claim
35 words
An adapted DNA information sequence having upstream a first adaptor molecule as in claim 1 with a start signal, and downstream a second adaptor molecule as in claim 1 with at least one stop signal.
Claim 23
Dependent Claim
20 words
The adapted DNA information sequence of claim 19 wherein at least one of the adaptors is a symmetrical DNA duplex.
Claim 24
Dependent Claim
10 words
The adapted DNA sequence of claim 20 comprising the structure:
Claim 25
Dependent Claim
48 words
The adapted DNA sequence of claim 21 wherein the start adaptor has the structure: where X and Y are nucleotide bases inserted to correct the frame shift, and Z is 1 or 2; or is the end product of digestion thereof with the enzyme for the recognition site.
Claim 26
Dependent Claim
27 words
The adapted DNA sequence of claim 19 with at least one additional nucleotide base between the start signal and the recognition site to correct the frame shift.
Claim 27
Dependent Claim
10 words
The adapted DNA sequence of claim 21. comprising the structure:
Claim 28
Dependent Claim
11 words
The adapted DNA sequence of claim 19 comprising the structure: -
Claim 29
Dependent Claim
23 words
The adapted DNA sequence of claim 23 ligated at both ends into a replicable cloning vehicle having the appropriate restriction enzyme recognition sites.
Claim 30
Dependent Claim
14 words
The adapted DNA sequence-cloning vehicle combination of claim 26 in a transformed host cell.
Claim 31
Dependent Claim
21 words
28 . A transformed host cell comprising as part of its genetic information sequences, an adapted DNA sequence of claim 23.
Claim 32
Dependent Claim
28 words
29 . The partial duplex DNA molecule, adapted for forming insulin A-chain gene, having the structure: 30. The duplex DNA molecule, gene for insulin A-chain, having the structure:
Claim 33
Dependent Claim
28 words
29 . The partial duplex DNA molecule, adapted for forming insulin A-chain gene, having the structure: 30. The duplex DNA molecule, gene for insulin A-chain, having the structure:
Claim 34
Dependent Claim
55 words
. The duplex DNA gene as in claim 30 including in addition adaptor DNA segments at each end, said adaptor segments including adjacent the upstream end of the gene a start signal, and adjacent the downstream end of the gene a stop signal, each adaptor segment also including a recognition site for a restriction endonuclease.
Claim 35
Dependent Claim
18 words
The duplex DNA of claim 31 wherein said' start signal is the ATG codon or the GTG codon.
Claim 36
Dependent Claim
22 words
The duplex DNA of claim 31 wherein the said stop signal is at least one of the codons TGA, TAA and TAG.
Claim 37
Dependent Claim
27 words
3 4. The duplex DNA of claim 31 wherein the recognition sites are selected from those selective for the endonucleases Eco RI, Bam HI, and Hin dIII.
Claim 38
Dependent Claim
30 words
The duplex DNA of claim 31 wherein the start signal adaptor contains an Eco RI site and comprises the structure: or the end product thereof after Eco RI enzyme digestion.
Claim 39
Dependent Claim
30 words
The duplex DNA of claim 31 wherein the stop signal adaptor contains a Bam HI site and has the structure: or the end product thereof after Bam HI enzyme digestion.
Claim 40
Dependent Claim
38 words
The duplex DNA molecule as in claim 31 wherein the two adaptors are in partial duplex form and both have protruding cohesive ends which are part of the recognition sites and capable of being rejoined to complementary sequences.
Claim 41
Dependent Claim
11 words
The adapted insulin A-chain gene of claim 37 comprising the structure:
Claim 42
Dependent Claim
13 words
The insulin A-chain gene of claim 37 ligated to a replicable cloning vehicle.
Claim 43
Dependent Claim
26 words
The gene-cloning vehicle hybrid of claim 39 wherein the cloning vehicle is a plasmid or a plasmid fragment resulting from cutting a plasmid with restriction endonuclease.
Claim 44
Dependent Claim
23 words
The hybrid of claim 40 wherein the plasmid is of the type: pBR322, pBGP120, or a hybrid of pBGP120 and p KN 402.
Claim 45
Dependent Claim
10 words
The hybrid of claim 39 in a transformed host cell.
Claim 46
Dependent Claim
9 words
Transformed bacterial cells containing the hybrid of claim 39.
Claim 47
Dependent Claim
14 words
Transformed bacterial cells of claim 43 which are a selected strain of E. coli.
Claim 48
Dependent Claim
18 words
' A culture of the transformed host cells of . claim 42, capable of producing insulin A-chain protein.
Claim 49
Dependent Claim
16 words
A culture of the transformed bacterial cells of claim 43, capable of producing insulin A-chain protein.
Claim 50
Dependent Claim
16 words
The partial duplex DNA molecule, adapted for forming a complete insulin B-chain gene, having the structure:
Claim 51
Dependent Claim
16 words
The partial duplex DNA molecule, adapted for forming a complete insulin B-chain gene, having the structure:
Claim 52
Dependent Claim
13 words
The duplex DNA molecule, gene for the B-chain of insulin, having the structure:
Claim 53
Dependent Claim
13 words
The duplex DNA molecule, gene for the B-chain of insulin, having the structure:
Claim 54
Dependent Claim
52 words
The duplex DNA gene as in claim 48 including adaptor DNA segments at each end, said adaptor segments including a start signal adjacent the upstream end of the gene, and a stop signal adjacent the downstream end of the gene, each adaptor segment also including a recognition site for a restriction endonuclease.
Claim 55
Dependent Claim
18 words
The duplex DNA of claim 49 wherein said start signal is the ATG codon or the GTG codon.
Claim 56
Dependent Claim
23 words
The duplex DNA of claim 49 wherein said stop signal is at least one codon selected from the group: TGA, TAA and TAG.
Claim 57
Dependent Claim
24 words
The duplex DNA of claim 49 wherein the recognition sites are'chosen from those selective for the endonucleases Eco RI, Bam HI, and Hin dIII.
Claim 58
Dependent Claim
24 words
The duplex DNA of claim 49 wherein the start signal adaptor has the structure: or the end product thereof after Eco RI enzyme digestion.
Claim 59
Dependent Claim
24 words
The duplex DNA of claim 49 wherein the stop signal adaptor has the structure: or the end product thereof after Bam HI enzyme digestion.
Claim 60
Dependent Claim
35 words
The DNA gene as in claim 49 wherein the two adaptors are in partial duplex form and both have protruding cohesive ends which are recognized partial recognition sites capable of being rejoined to complementary sequences.
Claim 61
Dependent Claim
13 words
The insulin B-chain gene of claim 55 with adaptor molecules having the structure:
Claim 62
Dependent Claim
13 words
The insulin B-chain gene of claim 55 ligated to a replicable cloning vehicle.
Claim 63
Dependent Claim
27 words
The B-chain gene-cloning vehicle hybrid of claim 57 wherein the cloning vehicle is a plasmid or a plasmid fragment resulting from cutting a plasmid with restriction endonuclease.
Claim 64
Dependent Claim
21 words
The hybrid of claim 58 wherein the plasmid is of the type: pBR322, pBGP120, or a hybrid of pBGP120 and pKN401.
Claim 65
Dependent Claim
10 words
The hybrid of claim 57 in a transformed host cell.
Claim 66
Dependent Claim
9 words
Transformed bacterial cells containing the hybrid of claim 57.
Claim 67
Dependent Claim
14 words
Transformed bacterial cells of claim 61 which are a selected strain of E. coli.
Claim 68
Dependent Claim
15 words
A culture of the transformed host cells of claim 60 for producing insulin B-chain protein.
Claim 69
Dependent Claim
15 words
A culture of the transformed bacterial cells of claim 61, for producing insulin B-chain protein.
Claim 70
Dependent Claim
64 words
, 65. In a method of inserting a genetic information sequence into a cloning vehicle and translating said sequence, the steps comprising enzymatically ligating a first start signal-containing adaptor molecule as in claim 1 to the upstream end of said genetic information sequence and ligating a second stop signal-containing adaptor molecule as in claim 1 to the downstream end of said genetic information sequence.
Claim 71
Dependent Claim
119 words
In a method.of inserting genetic information sequences into a cloning vehicle and translating said sequences, the steps comprising enzymatically ligating separate aliquot portions of said genetic information sequence separately to (a) a start signal-containing adaptor molecule as in claim 1, and (b) to at least one start signal-containing adaptor molecule as in claim 2, said start adaptors (b) containing at least one additional nucleotide base inserted between said start signal and the endonuclease recognition site to correct a possible inappropriate reading frame, thereby to form a multiplicity of adapted D N A sequences, inserting each of said adapted DNA sequences into a cloning vehicle and subsequently selecting 66.(cont.) an adapted DNA sequence which provides a desired product upon translation.
Claim 72
Dependent Claim
50 words
In a method of forming an insulin A-chain gene adapted for insertion into a cloning vehicle, the step of enzymatically ligating a start signal comprising one of: and proximate to the head end of an insulin A-chain gene whose 5' terminus at the tail end is in the 5'-OH form.
Claim 73
Dependent Claim
50 words
In a method of forming an insulin A-chain gene adapted for insertion into a cloning vehicle, the step of enzymatically ligating a start signal comprising one of: and proximate to the head end of an insulin A-chain gene whose 5' terminus at the tail end is in the 5'-OH form.
Claim 74
Dependent Claim
63 words
In a method of forming an insulin A-chain gene adapted for insertion into a cloning vehicle the step which comprises enzymatically reacting the 5'-OH tail end of the product of the process of claim 67 with ATP to convert the 5'-OH to 5'-OP and then enzymatically ligating a stop signal adaptor selected from the group consisting of: and to said 5'-OP tail end.
Claim 75
Dependent Claim
41 words
In a method of forming an insulin A-chain gene adapted for insertion into a cloning vehicle the step of enzymatically digesting the product of the process of claim 68 to form cohesive ends at both ends of said adapted A-chain gene.
Claim 76
Dependent Claim
66 words
A method of inserting an adapted insulin A-chain gene into a cloning vehicle which comprises enzymatically cutting and reacting a replicable DNA to form cohesive ends complimentary to the cohesive ends of the product of the process of claim 69 and then attaching each of the cohesive ends of said product of claim 69 to the corresponding complimentary cohesive ends of said enzymatically reacted replicable DNA.
Claim 77
Dependent Claim
51 words
In a method of forming an insulin B-chain gene adapted for insertion into a cloning vehicle, the step of enzymatically ligating a start signal adaptor comprising one of: and proximate to the head end of an insulin B-chain gene whose 5' terminus at the tail end is in the 5'-OH form.
Claim 78
Dependent Claim
51 words
In a method of forming an insulin B-chain gene adapted for insertion into a cloning vehicle, the step of enzymatically ligating a start signal adaptor comprising one of: and proximate to the head end of an insulin B-chain gene whose 5' terminus at the tail end is in the 5'-OH form.
Claim 79
Dependent Claim
64 words
In a method of forming an insulin B-chain gene adapted for insertion into a cloning vehicle the step which comprises enzymatically reacting the 5'-OH tail end of the product of the process of claim 71 with ATP to convert the S'-OH to 5'-OP and then enzymatically ligating a stop 72.(cont.) signal adaptor selected from the group consisting of and to said 5'-OP tail end.
Claim 80
Dependent Claim
42 words
In a method of forming an insulin B -chain gene adapted for insertion into a cloning vehicle the step of enzymatically digesting the product of the process fo claim 72 to form cohesive ends at both ends of said adapted B-chain gene.
Claim 81
Dependent Claim
66 words
A method of inserting an adapted insulin B-chain gene into a cloning vehicle which comprises enzymatically cutting and reacting a replicable DNA to form cohesive ends complimentary to the cohesive ends of the product of the process of claim 73 and then attaching each of the cohesive ends of said product of claim 73 to the corresponding complimentary cohesive ends of said enzymatically reacted replicable DNA.
Claim 82
Dependent Claim
55 words
A method of producing in vitro a particular genetically-produced organic material which comprises culturing transformed host cells containing a genetic sequence which produces said organic material, which sequences have been inserted into a host-cell-contained cloning vehicle by 75. (cont.) means of the adaptor molecules of claim 1, under conditions which promote said organic material formation.
Claim 83
Dependent Claim
42 words
A method of producing insulin A-chain protein which comprises culturing transformed host cells containing an insulin A-chain protein-producing genetic sequence inserted into a host-cell-contained cloning vehicle by means of the adaptor molecules of claim 1, under conditions which promote said protein formation.
Claim 84
Dependent Claim
42 words
A method of producing insulin B-chain protein which comprises culturing transformed host cells containing an insulin B-chain protein-producing genetic sequence inserted into a host-cell-contained cloning vehicle by means of the adaptor molecules of claim 1, under conditions which promote said protein formation.
References Cited (Backward Citations)
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