US4985359A
Expired / Lapsed
US
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Vectors and methods for the construction of cDNA libraries
Assignee: LLSE OBERBAUMER
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Filing: 1986-09-25
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Granted: 1991-01-15
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Expires: 2006-09-25
Abstract
Abstract Vectors useful in the construction of cDNA libraries and methods for constructing these vectors are disclosed. According to the invention, a vector includes a polylinker region which is cut to linearize the vector. Oligo(dC) tails having terminal dideoxynucleotides are added to the two 3' ends of the vector. One oligo(dC) tail is removed and replaced with oligo(dT) to which poly(A) mRNA is annealed. The first complementary DNA strand is synthesized and tailed with oligo(dG), which anneals with the remaining oligo(dC) tail, thus circularizing the vector. The mRNA is digested and the second cDNA strand synthesized. Alternatively, the second cDNA strand can be synthesized before circularizing the vector. The resulting vector is used to transform microorganisms to create a cDNA library. The cDNA can be recovered by cleaving the vector at sites within the polylinker sequence.
Inventors
- 👤 Ilse Oberbaumer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 03:38
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
152 words
A process for constructing a cDNA library comprising a. linearizing a vector containing a polylinker by treatment with restriction endonuclease, b. adding oligo(dC) or oligo(dG) tails to the 3' ends, c. adding dideoxynucleotide residues to the oligo(dC) or oligo(dG) tails, d. removing one of the oligo(dC) or oligo(dG) tails at the 3' end with a different restriction endonuclease, e. adding an oligo(dT) tail to the free 3' end, f. hybridizing linearized vector molecules via the oligo (dT) tail of step (e) to the poly(A) tail of mRNA molecules of a mRNA population, g. synthesizing double stranded cDNA from the mRNA molecules as template, h. adding an oligo(dC) or oligo(dG) tail to one of said cDNA strands, i. recircularizing said vector by annealing said oligo (dC) or oligo(dG) tail with a dideoxynucleotide-tailed complementary oligo(dG) or oligo(dC) tail on the other 3' end, and j. transforming a microorganism with the vector of step (i).
Claim 2
Dependent Claim
152 words
A process for constructing a cDNA library comprising a. linearizing a vector containing a polylinker by treatment with restriction endonuclease, b. adding oligo(dC) or oligo(dG) tails to the 3' ends, c. adding dideoxynucleotide residues to the oligo(dC) or oligo(dG) tails, d. removing one of the oligo(dC) or oligo(dG) tails at the 3' end with a different restriction endonuclease, e. adding an oligo(dT) tail to the free 3' end, f. hybridizing linearized vector molecules via the oligo (dT) tail of step (e) to the poly(A) tail of mRNA molecules of a mRNA population, g. synthesizing double stranded cDNA from the mRNA molecules as template, h. adding an oligo(dC) or oligo(dG) tail to one of said cDNA strands, i. recircularizing said vector by annealing said oligo (dC) or oligo(dG) tail with a dideoxynucleotide-tailed complementary oligo(dG) or oligo(dC) tail on the other 3' end, and j. transforming a microorganism with the vector of step (i).
Claim 3
Dependent Claim
13 words
A population of microorganisms containing a cDNA library constructed according to claim 1.
Claim 4
Dependent Claim
26 words
A linearized vector comprising an oligo(dT) tail at one 3' end and an oligo(dC) or oligo(dG) tail with a terminal dideoxynucleotide at the other 3' end.
Claim 5
Dependent Claim
26 words
A linearized vector comprising an oligo(dT) tail at one 3' end and an oligo(dC) or oligo(dG) tail with a terminal dideoxynucleotide at the other 3' end.
Claim 6
Dependent Claim
20 words
A linearized vector according to claim 3 comprising an oligo(dC) or oligo(dG) tail consisting of 20 cytidine or guanosine residues.
Claim 7
Dependent Claim
18 words
A linearized vector according to claim 3 comprising an oligo(dT) tail consisting of 60 to 100 thymidine residues.
Claim 8
Dependent Claim
72 words
A process for constructing a vector according to claim 3 comprising: a. linearizing a vector containing a polylinker by treatment with restriction endonuclease, b. adding oligo(dC) or oligo(dG) tails to the 3' ends, c. adding dideoxynucleotide residues to the oligo(dC) or oligo(dG) tails, d. removing one of the oligo(dC) or oligo(dG) tails at the 3' end with a different restriction endonuclease, and e. adding an oligo(dT) tail to the free 3' end.
Claim 9
Dependent Claim
29 words
A process according to claim 6 wherein said vector containing a polylinker is selected from the group consisting of plasmids pUC931, pUC830, pOM1, pOM2, pOM3, pOM4, pOM8, and pOM9.
Claim 10
Dependent Claim
3 words
The vector pUC931.
Claim 11
Dependent Claim
3 words
The vector pUC931.
Claim 12
Dependent Claim
12 words
A vector selected from the group consisting of: pOM1, pOM3 and pOM9.
Claim 13
Dependent Claim
12 words
A vector selected from the group consisting of: pOM1, pOM3 and pOM9.
Claim 14
Dependent Claim
3 words
The vector pUC830.
Claim 15
Dependent Claim
3 words
The vector pUC830.
Claim 16
Dependent Claim
12 words
A vector selected from the group consisting of: pOM2, pOM4 and pOM8.
Claim 17
Dependent Claim
12 words
A vector selected from the group consisting of: pOM2, pOM4 and pOM8.
References Cited (Backward Citations)
US4394443A
Cited
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