Recombinant methods and materials

Claim 1

A method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome, said method comprising: (1) isolating from said genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following said site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) said first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of said fragment, and (b) said second altered fragment comprising the fragment having said selected DNA sequence inserted therein or deleted therefrom; (3) contacting said genome with said first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising said thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with said second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking said thymidine kinase gene, and isolating the recombinant genome product.

Claim 2

A method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome, said method comprising: (1) isolating from said genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following said site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) said first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of said fragment, and (b) said second altered fragment comprising the fragment having said selected DNA sequence inserted therein or deleted therefrom; (3) contacting said genome with said first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising said thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with said second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking said thymidine kinase gene, and isolating the recombinant genome product.

Claim 6

The method according to claims 1 or 4 wherein the step of selecting for a recombinant genome not comprising said thymidine kinase gene comprises the step of exposing the recombinant genome to a growth medium comprising AraT.