Methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins
First Claim
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1. A method for preparing a nucleic acid, the method comprising the steps of:
- (i) amplifying a nucleic acid that encodes a polypeptide using a primer complementary to at least part of a synthetic sequence located at the 5′
terminus of the nucleic acid sequence;
(ii) rendering the amplified nucleic acid obtained from step (i) single-stranded;
(iii) hybridizing the single-stranded nucleic acid obtained from step (ii) with a single-stranded oligonucleotide to form a locally double-stranded region, wherein there is a single site for a restriction endonuclease within the locally double-stranded region;
(iv) forming a cleaved DNA complex by cleaving the locally double-stranded region formed by the hybridizing step (iii) with the restriction endonuclease at the single site for the restriction endonuclease to remove all unwanted 5′
nucleotides from the amplified nucleic acid; and
(v) cloning the cleaved DNA complex obtained from step (iv) a vector for expressing the polypeptide encoded by the nucleic acid;
wherein the hybridizing and the cleaving steps are performed at a temperature under which the single-stranded nucleic acid is maintained in substantially single-stranded form and associates with the single-stranded oligonucleotide to form the locally double-stranded region, and wherein the restriction endonuclease is active at the temperature.
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Abstract
Methods useful in constructing libraries that collectively display members of diverse families of peptides, polypeptides or proteins and the libraries produced using those methods. Methods of screening those libraries and the peptides, polypeptides or proteins identified by such screens.
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Citations
27 Claims
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1. A method for preparing a nucleic acid, the method comprising the steps of:
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(i) amplifying a nucleic acid that encodes a polypeptide using a primer complementary to at least part of a synthetic sequence located at the 5′
terminus of the nucleic acid sequence;(ii) rendering the amplified nucleic acid obtained from step (i) single-stranded; (iii) hybridizing the single-stranded nucleic acid obtained from step (ii) with a single-stranded oligonucleotide to form a locally double-stranded region, wherein there is a single site for a restriction endonuclease within the locally double-stranded region; (iv) forming a cleaved DNA complex by cleaving the locally double-stranded region formed by the hybridizing step (iii) with the restriction endonuclease at the single site for the restriction endonuclease to remove all unwanted 5′
nucleotides from the amplified nucleic acid; and(v) cloning the cleaved DNA complex obtained from step (iv) a vector for expressing the polypeptide encoded by the nucleic acid; wherein the hybridizing and the cleaving steps are performed at a temperature under which the single-stranded nucleic acid is maintained in substantially single-stranded form and associates with the single-stranded oligonucleotide to form the locally double-stranded region, and wherein the restriction endonuclease is active at the temperature. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Current AssigneeTakeda Pharmaceutical Company Limited
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Original AssigneeDyax Corporation (Shire plc)
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InventorsRookey, Kristin L., Ladner, Robert C., Cohen, Edward H., Nastri, Horacio G., Hoet, Rene
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Primary Examiner(s)Crow, Robert T.
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Assistant Examiner(s)Dauner, Joseph G
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Application NumberUS11/365,556Publication NumberTime in Patent Office3,780 DaysField of SearchNoneUS Class Current1/1CPC Class CodesC12N 15/10 Processes for the isolation...C12N 15/1037 Screening libraries present...C12N 15/1093 General methods of preparin...C12N 15/66 General methods for inserti...C40B 40/02 Libraries contained in or d...
