Procedure for the polymerization of nucleic acid sequences and i

Details Procedure for the polymerization of nucleic acid sequences and i Procedure for the polymerization of nucleic acid sequences and i

1996-09-16

1999-01-26

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 691, 435 911, 4351723, 536 221, 536 231, 536 243, 536 2431, 536 2432, 536 2433, C12Q 168, C12P 2100, C12N 1563, C07H 2104

Patent

active

058637303

ABSTRACT:
This procedure comprises the following steps: (1) cleavage of a circular cloning vector comprising two restriction sites a and b giving cohesive ends which are compatible with one another, and two restriction sites c and d which are close to the restriction sites a and b, by restriction enzyme(s), (2) introduction by ligation of the nucleic acid sequence to be polymerized into the linearized vector obtained in (1), between the two restriction sites a and b (1x vector), (3) cleavage of the recircularized vector obtained in (2), by restriction enzyme(s), (4) introduction by ligation of a gene coding for a suppressor tRNA into the linearized vector obtained in (3), between the two restriction sites c and d (1x+s vector), (5) cleavage of the vector obtained in (4) by restriction enzyme(s), (6) introduction by ligation of the fragment a-d obtained in (5) (1x+s fragment) between the sites b and d of a 1x vector as obtained in (2), after cleavage of this 1x vector at the said sites b and d, (7) introduction by ligation of the fragment obtained in (6) (2x+s fragment) between the sites b and d of a 1x vector as obtained in (2), after cleavage of this 1x vector, the cleavage being as defined in (6), (8) repetition of step (7), by insertion of the nx+s fragment into a 1x vector, until a sequence containing n+1 fragments is obtained, (9) transformation of a bacterial strain with the vector obtained in (8), and selection of mut+ strains, and (10) extraction of the (n+1)x polymer from the said selected strains.

REFERENCES:
patent: 4403036 (1983-09-01), Hartley et al.
patent: 5093251 (1992-03-01), Richards et al.
Molecular Cloning, A Laboratory Manual, Second Edition, 1989, pp. 1.6 and 1.19, J. Sambrook, et al., "Plasmid Vectors".
Lobel et al, "Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations", Proc. Natl. Acad. Sci. 81:4149-4153, Jul. 1984.
Taylor et al, "A general method for cloning DNA fragments in multiple copies", Gene 53:139-144, 1987.
Graham et al, "A rapid and reliable method to create tandem arrays of short DNA sequences", Biotechniques 13(5):780-789, 1992.
Hwang et al, "A method of cloning multiple direct repeats of a DNA segment", Biotechniques 14(5):766-767, 1993.

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