DNA-methylase linking reaction

Details DNA-methylase linking reaction DNA-methylase linking reaction

1994-09-09

1999-01-05

Chambers, Jasemine C.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 71, 435 697, 4351721, 4353201, 536 231, C12Q 168, G01N 3353, C12N 1509, C07H 2104

Patent

active

058560904

ABSTRACT:
The activity of sequence-specific DNA binder proteins, such as DNA methylases, provides a method of obtaining a covalent linkage between a nucleic acid segment and a polypeptide determinant encoded by the nucleic acid segment. The polypeptide determinant is expressed as a fusion protein together with the DNA methylase, which binds in vivo to a cytidine suicide analog when present in a nucleotide sequence. A plasmid suitable for use in this linkage reaction can comprise: (1) a gene fusion construct including a gene encoding a DNA methylase and a gene encoding a polypeptide determinant; (2) a promoter for transcription of the gene fusion construct as messenger RNA; and (3) a methylase conjugation element linked to the gene fusion sequence, the methylase conjugation element including a methylase binding site having at least one copy of a nucleotide sequence including a cytidine suicide analog capable of irreversibly binding the DNA methylase. The plasmid can form a plasmid-polypeptide determinant conjugate. The plasmids and methods of the present invention are useful for in vitro evolution of proteins.

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