US4716105A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Mini Mu containing plasmid and a method for rapid DNA sequencing
Assignee: United States Department of Health and Human Services
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Filing: 1984-12-13
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Granted: 1987-12-29
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Expires: 2004-12-13
Abstract
Abstract The present invention discloses a rapid method of sequencing a relatively large segment of deoxyribonucleic acid. The method in part comprises high frequency insertion of a suitable transposon into a segment of DNA of interest. Preferable use of Mu transposons is described. A plasmid having mini-Mu transposons has been prepared and disclosed.
Inventors
- 👤 Kiyoshi Mizuuchi
- 👤 Michiyo Mizuuchi
- 👤 Toshiro Adachi
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 10:30
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C12N; C12N15/00
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
16 words
A plasmid having a mini Mu transposon of less than 2 kilobases inserted in plasmid DNA.
Claim 2
Dependent Claim
16 words
A plasmid having a mini Mu transposon of less than 2 kilobases inserted in plasmid DNA.
Claim 3
Dependent Claim
10 words
The plasmid of claim 1 being incorporated in ATCC 39943.
Claim 4
Independent Claim
130 words
A method of determining base sequence in a DNA segment comprising: (a) cloning said DNA segment by incorporation thereof in a cloning vector selected from the group consisting of ColE1 plasmids and M13 DNA phages; (b) randomly inserting a mini Mu marker transposon of less than 2 kilobases in cloned DNA of step (a); (c) isolating transposon-containing DNA obtained through step (b); (d) separating radiolabelled chain terminator reaction products obtained by using radiolabelled nucleotides as precursors and the DNA of step (c) as a template together with universal primer oligonucleotide having sequence complimentary to one of the transposon ends or two oligonucleotides each complimentary to each of the two ends of the tramsposon; and (e) identifying the sequence of said bases from said radiolabelled products through suitable gel elctrophoretic means.
Claim 5
Independent Claim
130 words
A method of determining base sequence in a DNA segment comprising: (a) cloning said DNA segment by incorporation thereof in a cloning vector selected from the group consisting of ColE1 plasmids and M13 DNA phages; (b) randomly inserting a mini Mu marker transposon of less than 2 kilobases in cloned DNA of step (a); (c) isolating transposon-containing DNA obtained through step (b); (d) separating radiolabelled chain terminator reaction products obtained by using radiolabelled nucleotides as precursors and the DNA of step (c) as a template together with universal primer oligonucleotide having sequence complimentary to one of the transposon ends or two oligonucleotides each complimentary to each of the two ends of the tramsposon; and (e) identifying the sequence of said bases from said radiolabelled products through suitable gel elctrophoretic means.
Claim 6
Dependent Claim
16 words
The method of claim 3 wherein said transposon has two end sequences of Mu phage DNA.
Claim 7
Dependent Claim
13 words
The method of claim 4 wherein said transposon has a drug resistance marker.
Claim 8
Dependent Claim
19 words
The method of claim 5 employing suitable Escherichia coli strains as a vehicle for phage multiplication and transposition reactions.
Claim 9
Dependent Claim
14 words
The method of claim 6 wherein said electrophoretic means is denaturing acrylamide gel electrophoresis.
Claim 10
Dependent Claim
17 words
The method of claim 7 wherein radiolabelled products are identified by autoradiographic means, optionally employing computer means.
Claim 11
Independent Claim
118 words
A method of sequencing a DNA segment comprising: (a) cloning said DNA segment in a M13 phage and preparing a stock of said phage having said DNA cloned therein; (b) infecting with said phage stock a suitable Escherichia coli strain having plasmid carrying mini Mu transposons and ampicillin resistant marker therein; (c) selecting ampicillin resistant phage particles having said transposons randomly incorporated in the cloned DNA during step (b); (d) extracting substantially purified DNA from said ampicillin resistant phage particles and employing said purified DNA as a template in a suitable chain terminator reaction using radiolabelled DNA precursors to obtain radiolabelled products; and (e) identifying base sequence from said radiolabelled products by gel electrophoretic, autoradiographic and computer means.
Claim 12
Independent Claim
118 words
A method of sequencing a DNA segment comprising: (a) cloning said DNA segment in a M13 phage and preparing a stock of said phage having said DNA cloned therein; (b) infecting with said phage stock a suitable Escherichia coli strain having plasmid carrying mini Mu transposons and ampicillin resistant marker therein; (c) selecting ampicillin resistant phage particles having said transposons randomly incorporated in the cloned DNA during step (b); (d) extracting substantially purified DNA from said ampicillin resistant phage particles and employing said purified DNA as a template in a suitable chain terminator reaction using radiolabelled DNA precursors to obtain radiolabelled products; and (e) identifying base sequence from said radiolabelled products by gel electrophoretic, autoradiographic and computer means.
Claim 13
Dependent Claim
13 words
The plasmid of claim 1 wherein said mini mu has drug resistant markers.
Claim 14
Dependent Claim
20 words
The method of claim 3 determining the base sequence from inside toward outside of the DNA segment of step (a).
Claim 15
Dependent Claim
14 words
The method of claim 3 completing the entire process in less than 96 hours.
References Cited (Backward Citations)
No citation records indexed.
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-12-28 | 1991-06-29 | 1991-12-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-12-28 | 1995-06-29 | 1995-12-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-12-28 | 1999-06-29 | 1999-12-29 | $7700.00 | Window Closed / Lapsed |