Oligonucleotide population preparation
First Claim
1. A method for producing a sequence tag from a polynucleotide, said method comprising:
- (a) digesting said polynucleotide with a type II restriction enzyme to produce a first cohesive end;
(b) providing a promoter-linker cassette comprising an RNA polymerase promoter, a restriction site for a type IIs restriction enzyme and a second cohesive end which is complementary to said first cohesive end produced by said type II restriction enzyme;
(c) ligating the digested polynucleotide to the cassette by joining said first cohesive end and said second cohesive end;
(d) digesting the ligated product of step (c) with said type IIs restriction enzyme; and
(e) transcribing the resulting construct from said promoter, thereby producing a sequence tagged polyribonucleotide.
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Abstract
Methods are disclosed for producing a sequence tag from a polynucleotide. In preferred embodiments, the method comprises (a) digesting the polynucleotide with a type II restriction enzyme to produce a first cohesive end; (b) providing a promoter-linker cassette comprising an RNA polymerase promoter, a restriction site for a type IIS restriction enzyme and a second cohesive end which is complementary to the first cohesive end produced by the type II restriction enzyme; (c) ligating the digested polynucleotide to the cassette by joining the first cohesive end and the second cohesive end; (d) digesting the ligated product of step (c) with the type Iis restriction enzyme; and (e) transcribing the resulting construct from the promoter thereby producing a sequence tagged polyribonucleotide.
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Citations
17 Claims
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1. A method for producing a sequence tag from a polynucleotide, said method comprising:
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(a) digesting said polynucleotide with a type II restriction enzyme to produce a first cohesive end; (b) providing a promoter-linker cassette comprising an RNA polymerase promoter, a restriction site for a type IIs restriction enzyme and a second cohesive end which is complementary to said first cohesive end produced by said type II restriction enzyme; (c) ligating the digested polynucleotide to the cassette by joining said first cohesive end and said second cohesive end; (d) digesting the ligated product of step (c) with said type IIs restriction enzyme; and (e) transcribing the resulting construct from said promoter, thereby producing a sequence tagged polyribonucleotide. - View Dependent Claims (2, 6, 7, 9, 10, 11, 12)
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3. A promoter-linker cassette oligonucleotide comprising an RNA polymerase promoter which is situated 5'"'"' to a restriction site for a type IIS restriction enzyme which is situated 5'"'"' to a restriction site for a type II restriction enzyme.
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4. A method for producing a sequence tag from a polynucleotide, said method comprising:
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(a) digesting said polynucleotide with a type II restriction enzyme to produce a first cohesive end; (b) providing a promoter-linker cassette comprising a polymerase promoter, a restriction site for a type IIs restriction enzyme and a second cohesive end which is complementary to said first cohesive end produced by said type II restriction enzyme; (c) ligating the digested polynucleotide to the cassette; (d) digesting the ligated product of step (c) with said type IIs restriction enzyme; (e) circularizing the product of step (d); (f) digesting the circularized product with said type II restriction enzyme; and (g) transcribing the resulting construct, thereby producing a sequence tagged polynucleotide. - View Dependent Claims (5, 8)
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13. A method for producing a sequence tag from a polynucleotide, said method comprising:
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(a) shearing said polynucleotide; (b) providing a promoter-linker cassette comprising an RNA polymerase promoter and a restriction site for a type IIs restriction enzyme; (c) blunting said polynucleotide; (d) ligating the sheared polynucleotide to the cassette; (e) digesting the ligated product of step (d) with said type IIs restriction enzyme; and (f) transcribing the resulting construct from said promoter, thereby producing a sequence tagged polyribonucleotide.
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14. A method for producing a unique sequence tag from a polynucleotide, said method comprising:
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(a) digesting said polynucleotide with a type II restriction enzyme; (b) ligating the digested polynucleotide to a promoter-linker cassette comprising an RNA polymerase promoter and a cohesive end complementary to cohesive ends produced by said type II restriction enzyme, such that the ligated product contains a recognition site for a type IIs restriction enzyme; (c) digesting the ligated product of step (b) with said type IIs restriction enzyme; and (d) transcribing the resulting construct from said promoter, thereby producing a sequence tagged polyribonucleotide. - View Dependent Claims (15)
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16. A method for producing a sequence tag from a polynucleotide, said method comprising:
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(a) digesting said polynucleotide with a type II restriction enzyme to produce a first cohesive end; (b) providing a promoter-linker cassette comprising an RNA polymerase promoter, a restriction site for a type IIs restriction enzyme and a second cohesive end which is complementary to said first cohesive end produced by said type II restriction enzyme, wherein said cassette is digested to produce said second cohesive end; (c) ligating the digested polynucleotide to the cassette by joining said first cohesive end and said second cohesive end; (d) digesting the ligated product of step (c) with said type IIs restriction enzyme; (e) blunting the ends of the product of the type IIs digestion; and (f) transcribing the resulting construct from said promoter, thereby producing a sequence tagged polyribonucleotide.
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17. A method for producing a sequence tag from a polynucleotide, said method comprising:
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(a) digesting said polynucleotide with a type II restriction enzyme to produce a first cohesive end; (b) providing a promoter-linker cassette comprising an RNA polymerase promoter, a restriction site for a type IIs restriction enzyme and a second cohesive end which is complementary to said first cohesive end produced by said type II restriction enzyme, wherein said cassette is digested to produce said second cohesive end; (c) ligating the digested polynucleotide to the cassette; (d) digesting the ligated product of step (c) with said type IIs restriction enzyme; (e) blunting the ends of the product of the type IIs digestion; (f) circularizing the product of step (e); (g) digesting the circularized product with said type II restriction enzyme; (h) blunting the ends of the product of the type II digestion; and (i) transcribing the resulting construct from said promoter, thereby producing a sequence tagged polyribonucleotide.
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Specification