EP0530112A2
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EP
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Method for synthesizing singlestranded stem-loop DNAS, the products and uses therefor
Assignee: Rutgers, The State University of New Jersey
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Filing: 1992-08-28
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Granted: —
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Expires: 2012-08-28
Abstract
Abstract The invention conerns the field of recombinant DNA. More particularly, the invention relates to a method of synthesis of new and useful single-stranded DNA which have a stem-loop configuration (ss-slDNA). The invention relates to an in vitro and in vivo method of synthesis. Further, the invention relates to the replicating vehicle which produce these ss-slDNAs. Moreover, the invention relates these novel structures and discloses uses for these structures. There is described a method for amplifying ss-slDNAs with or without genes encoding a target protein.
Inventors
- 👤 Atsushi Ohshima
- 👤 Sumiko Inouye
- 👤 Masayori Inouye
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 11:02
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/10; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2468; C12N9/2471; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; C12N2310/00; C12N2310/10; C12N2310/11; C12N2310/111; C12N2310/15; C12N2310/50; C12N2310/53
Claims Analysis
Total Claims: 36
Claim 1
Independent Claim
246 words
A method for synthesizing a novel single-stranded DNA (ssDNA) molecule which comprises a stem-loop structure (slDNA) which stem is constituted of duplexed DNA of annealed complementary bases within the ssDNA, which stem forms, at one end, the 5′ and 3′ termini of the ssDNA molecule and at the other end, a single-stranded loop of DNA joining the opposite ends of the duplexed DNA in a system which contains the necessary components of DNA polymerase synthesis, and which system comprises: (a) a template DNA containing a suitable priming site and an inverted repeat (IR) downstream of said priming site, (b) a primer for the template to allow the start of DNA polymerization, and (c) a DNA polymerase to replicate the slDNA from the template, which method comprises the steps of: (1) priming the template to allow the start of DNA polymerization, (2) synthesizing the ssDNA from the primer using one of the double-strand of the DNA as template, continuing the DNA synthesis into the IR sequence and allowing the synthesis to cease within the IR sequence, (3) allowing complementary bases within the newly synthesized strand at the IR sequence to anneal forming a loop of a non-duplexed region and a duplexed region at the neck of the loop, the duplexed region functioning as a priming site for continued DNA synthesis, (4) resuming DNA synthesis using as template the newly synthesized strand and/or the other strand of the DNA, (6) forming the slDNA, and (5) separating the slDNA.
Claim 2
Dependent Claim
30 words
The method of claim 1 wherein the DNA synthesis after formation of the loop, is allowed to continue until the second strand is longer with respect to the first strand.
Claim 3
Dependent Claim
28 words
The method of claim 1 wherein after formation of the loop, synthesis of the second strand is stopped before the second strand is longer than the first strand.
Claim 4
Dependent Claim
22 words
The method of claim 1 wherein the system includes two polymerases, one synthesizing the first strand and one synthesizing the second strand.
Claim 5
Dependent Claim
20 words
The method of claim 1 wherein the template for the synthesis of the second strand is the first synthesized strand.
Claim 6
Dependent Claim
24 words
The method of claim 1 wherein the template for the synthesis of the second strand is the other strand of the original template DNA.
Claim 7
Dependent Claim
16 words
The method of claim 1 wherein the synthesis is carried out in a self-replicating DNA vehicle.
Claim 8
Dependent Claim
12 words
The method of claim 7 wherein the self-replicating vehicle is a plasmid.
Claim 9
Dependent Claim
13 words
The method of claim 8 wherein the plasmid is an E. coli plasmid.
Claim 10
Dependent Claim
10 words
The method of claim 9 wherein the plasmid is pUCK106.
Claim 11
Dependent Claim
25 words
The method of claim 10 wherein the plasmid includes a gene for encoding a protein positioned between the origin of replication and the inverted repeats.
Claim 12
Dependent Claim
13 words
The method of claim 11 wherein the gene is the lac Z gene.
Claim 13
Dependent Claim
23 words
The method of claim 12 wherein the plasmid has an origin of replication for the first polymerase positioned upstream of the inverted repeats.
Claim 14
Dependent Claim
21 words
The method of claim 1 wherein there is produced a family of slDNAs wherein the respective 5′ ends vary in size.
Claim 15
Dependent Claim
12 words
The method of claim 1 which is carried out in vitro .
Claim 16
Dependent Claim
22 words
The method of claim 15 which includes two polymerases: one to synthesize the first strand and one to synthesize the second strand.
Claim 17
Dependent Claim
9 words
The slDNA obtained by the method of claim 1.
Claim 18
Dependent Claim
9 words
The slDNA obtained by the method of claim 10.
Claim 19
Independent Claim
131 words
A system for synthesizing a novel single-stranded DNA (ssDNA) molecule which comprises a stem-loop structure (slDNA) which stem is constituted of duplexed DNA of annealed complementary bases within the ssDNA and forms at one end, the 5′ and 3′ termini of the ssDNA molecule and at the other end, a single-stranded loop of DNA joining the opposite ends of the duplexed DNA, which system also contains the necessary components of DNA polymerase synthesis, and which system comprises: (a) a template DNA fragment containing a suitable priming site and an inverted repeat (IR) downstream of said priming site, (b) a primer for the template to allow the start of DNA polymerization, and (c) a DNA polymerase to replicate the slDNA from the template and/or from the second strand of the DNA fragment.
Claim 20
Dependent Claim
25 words
The system of claim 19 which includes two polymerases, one for synthesizing the first strand of the ssDNA and one for synthesizing the second strand.
Claim 21
Dependent Claim
19 words
The system of claim 20 which is an in vivo system which also includes a self-replicating competent DNA vehicle.
Claim 22
Dependent Claim
13 words
The system of claim 21 wherein the self-replicating DNA vehicle is a plasmid.
Claim 23
Dependent Claim
13 words
The system of claim 22 wherein the plasmid is an E. coli plasmid.
Claim 24
Dependent Claim
10 words
The system of claim 23 wherein the plasmid is pUCK106.
Claim 25
Dependent Claim
30 words
The system of claim 24 wherein the plasmid also contains a DNA sequence capable of encoding a protein, which is located between the origin of replication and the inverted repeats.
Claim 26
Dependent Claim
12 words
The system of claim 25 wherein the DNA sequence is a gene.
Claim 27
Dependent Claim
13 words
The system of claim 26 wherein the gene is the lac Z gene.
Claim 28
Dependent Claim
62 words
An isolated and purified novel single-stranded DNA (ssDNA) molecule which comprises a stem-loop structure (slDNA) which stem is constituted of duplexed DNA of annealed complementary bases within the ssDNA, which stem forms, at one end, the 5′ and 3′ termini of the ssDNA molecule and at the other end, a single-stranded loop of DNA joining the opposite ends of the duplexed DNA.
Claim 29
Dependent Claim
26 words
The novel single-stranded DNA (ssDNA) of claim 28 wherein either the 3′ or the 5′ end of the stem is longer with respect to the other.
Claim 30
Dependent Claim
20 words
The novel single-stranded DNA (ssDNA) of claim 29 wherein the 3′ end is the longer one of the two ends.
Claim 31
Dependent Claim
17 words
A family of slDNAs of claim 28 wherein the respective 5′ end single-stranded sequence vary in size.
Claim 32
Dependent Claim
19 words
The slDNA of claim 28 which was synthesized from a template DNA which included a 35-bp inverted repeat sequences.
Claim 33
Dependent Claim
12 words
The slDNA of claim 28 wherein the single-stranded loop comprises four bases.
Claim 34
Dependent Claim
30 words
The novel single-stranded DNA (ssDNA) of claim 28 which comprises a DNA sequence capable of encoding a protein, which is located between the loop and the terminus of the stem.
Claim 35
Dependent Claim
15 words
The novel single-stranded DNA (ssDNA) of claim 34 wherein the DNA sequence is a gene.
Claim 36
Dependent Claim
15 words
The novel single-stranded DNA (ssDNA) of claim 35 wherein the gene is that encoding β-galactosidase.
References Cited (Backward Citations)
No citation records indexed.
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