Random Mutagenesis And Amplification Of Nucleic Acid

a nucleic acid and mutagenesis technology, applied in combinational chemistry, dna preparation, library creation, etc., can solve the problems of tertiary structure, biological function remains elusive, and formidable tasks, and achieve the goal of promoting heterologous binding of oligonucleotides, enhancing degeneracy of oligonucleotides, and increasing the efficiency of extension of oligonucleotides

Inactive Publication Date: 2008-09-25
GENOPSYS
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Benefits of technology

[0057]The library of oligonucleotides may optionally be synthetic and may be synthesized by randomly incorporating A, T, G, C, I or U. Optionally, at least one of the oligonucleotides used in the library of oligonucleotide in the above methods has one or more inosine residues at the 3′ end of the oligonucleotide, preferably 1-5 inosine residues, more prefera

Problems solved by technology

However, to a large extent, the correlation between protein primary sequence, tertiary structure, and biological function remains elusive.
Although it is desirable to be able to predict protein folding pattern from its primary sequence and to correlate it

Method used

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  • Random Mutagenesis And Amplification Of Nucleic Acid
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  • Random Mutagenesis And Amplification Of Nucleic Acid

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[0166]The gene encoding a penicillinase from Bacillus licheniformis was used as a target to be randomly mutagenized. By randomly mutating the enzyme, isozymes which show altered hydrolytic activity and / or specificity against various penicillins and cephalosporins may offer clues to 1) how antibiotics can be designed to thwart the inevitable evolution towards β-lactamases which render pathogenic bacteria resistant to drug therapy, and 2) offer further information for the study of protein structure-function relationships.

[0167]The gene encoding the Bacillus licheniformis was isolated from a plasmid pELB1. The plasmid pELB1 is a pBR322 derivative, containing the “exolarge” form of the B. licheniformis b-lactamase gene, utilizing the Bacillus amyloliquefaciens promoter and subtilisin signal sequence, and Bacillus and E. coli origins of replication (Ellerby, L. M., Escobar, W. A., Fink, A. L., Mitchinson C., Wells J A (1990) Biochemistry, June 19; 29(24):5797-806).

[0168]pELB1 was digeste...

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Abstract

A method is provided for mutagenizing nucleic acids and proteins relative to an initial nucleic acid sequence by the insertion, deletion or substitution of nucleotide(s) in the target nucleic acid during amplification.

Description

CROSS-REFERENCE[0001]This application is a divisional application of application Ser. No. 10 / 069,442, filed Jun. 28, 2008, which is a 371 of PCT / US00 / 22078, filed on Aug. 11, 2000, which is a continuation-in-part of application Ser. No. 09 / 374,274, filed on Aug. 13, 1999, now U.S. Pat. No. 6,251,604, which are incorporated herein by reference in their entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to methods for mutagenizing nucleic acids and proteins. More particularly, the present invention relates to methods for mutagenizing nucleic acids and proteins relative to an initial target nucleic acid sequence by the insertion, deletion, or substitution of nucleotide(s) in the target nucleic acid during amplification.[0004]2. Description of Related Art[0005]The sequences of genes encoding many important proteins have been determined at a rapid speed owing to the fast progress in the field of genomics. The three-dimensional structure...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N9/86C12N15/10
CPCC12N15/1093C12N15/102
Inventor LIETZ, ERIC
Owner GENOPSYS
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