US4987073A
Expired / Lapsed
US
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Bacteriophage cloning system for the construction of directional DNA libraries
Assignee: Akzo NV
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Filing: 1990-03-14
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Granted: 1991-01-22
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Expires: 2010-03-14
Abstract
Abstract A bacteriophage directional cloning vector containing a multiple cloning site, a method for preparing cDNAs that can be cloned in a unique orientation and a bifunctional oligodeoxynucleotide linker, which creates a unique restriction site when ligated to the 3' end of cDNA fragments. The simple and efficient cDNA cloning system reduces the amount of RNA and effort required for the preparation of large directionally cloned libraries by providing a unique vector that accepts cDNA fragments in a unique 5'-3' orientation and by providing a unique bifunctional linker that facilitates the preparation of cDNA fragments that can be ligated directionally into the vector.
Inventors
- 👤 Michael L. Berman
- 👤 P. Scott Meissner
- 👤 William P. Sisk
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 20:19
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/66; C12N15/74
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
138 words
A highly efficient method for modifying and unidirectionally inserting cDNA into a cloning vector, comprising: synthesizing a double stranded oligonucleotide linker comprising a complete restriction enzyme recognition site joined to nucleotides comprising a different and incomplete restriction enzyme recognition site, such sites being in a 5'-incomplete-complete-3' configuration; ligating the linker to both ends of a double stranded cDNA fragment to form a cDNA-linker composition such that an incomplete restriction enzyme recognition site is reconstituted to a complete restriction enzyme site only at the 3' poly A end of the cDNA fragment; cleaving said cDNA-linker composition with two different restriction enzymes, said enzymes being specific for said restriction sites resulting in a cDNA with two asymmetrical restriction enzyme recognition sites; and inserting unidirectionally the cleaved cDNA with asymmetrical restriction enzyme recognition sites into a vector containing complementary asymmetrical sites.
Claim 2
Dependent Claim
138 words
A highly efficient method for modifying and unidirectionally inserting cDNA into a cloning vector, comprising: synthesizing a double stranded oligonucleotide linker comprising a complete restriction enzyme recognition site joined to nucleotides comprising a different and incomplete restriction enzyme recognition site, such sites being in a 5'-incomplete-complete-3' configuration; ligating the linker to both ends of a double stranded cDNA fragment to form a cDNA-linker composition such that an incomplete restriction enzyme recognition site is reconstituted to a complete restriction enzyme site only at the 3' poly A end of the cDNA fragment; cleaving said cDNA-linker composition with two different restriction enzymes, said enzymes being specific for said restriction sites resulting in a cDNA with two asymmetrical restriction enzyme recognition sites; and inserting unidirectionally the cleaved cDNA with asymmetrical restriction enzyme recognition sites into a vector containing complementary asymmetrical sites.
Claim 3
Dependent Claim
13 words
A method according to claim 1, wherein said oligonucleotide linker is single stranded.
Claim 4
Dependent Claim
22 words
A method according to claim 2, wherein the complete site is a BamHl restriction site bound symmetrically by the nucleotide sequence GCTT.
Claim 5
Dependent Claim
17 words
A method according to claim 3, wherein said reconstituted restriction enzyme recognition site is to HindIII endonuclease.
Claim 6
Dependent Claim
17 words
A method according to claim 1, wherein said two restriction enzymes are BamHl endonuclease and HindIII endonuclease.
Claim 7
Dependent Claim
12 words
A method according to claim 1, wherein said vector is lambda ORF8.
Claim 8
Dependent Claim
79 words
A composition consisting essentially of a double stranded cDNA with a ply A end and a double stranded linker which linker contains a complete restriction enzyme recognition site flanked on both ends by a different and incomplete restriction enzyme recognition site which incomplete site contains nucleotides that allow the formation of a complete restriction enzyme recognition site only at the poly A end of the cDNA when the linker and cDNA are incubated under conditions that allow intermolecular ligation.
Claim 9
Dependent Claim
79 words
A composition consisting essentially of a double stranded cDNA with a ply A end and a double stranded linker which linker contains a complete restriction enzyme recognition site flanked on both ends by a different and incomplete restriction enzyme recognition site which incomplete site contains nucleotides that allow the formation of a complete restriction enzyme recognition site only at the poly A end of the cDNA when the linker and cDNA are incubated under conditions that allow intermolecular ligation.
Claim 10
Dependent Claim
37 words
A linker-cDNA composition according to claim 7, wherein said incomplete restriction enzyme recognition site is the partial HindIII restriction site nucleotide sequence of GCTT and said complete restriction enzyme recognition site is the complete BamHl restriction site.
Claim 11
Dependent Claim
18 words
A linker-cDNA composition according to claim 7, wherein said reconstituted restriction enzyme recognition site is to HindIII endonuclease.
Claim 12
Dependent Claim
13 words
A linker-cDNA composition according to claim 17, wherein said vector is lambda ORF8.
References Cited (Backward Citations)
US4666839A
Cited
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-01-21 | 1994-07-23 | 1995-01-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-01-21 | 1998-07-23 | 1999-01-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-01-21 | 2002-07-23 | 2003-01-22 | $7700.00 | Window Closed / Lapsed |