Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes.

ABSTRACT: We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retr

SUBMITTER: Hobom U 

PROVIDER: S-EPMC112300 | biostudies-literature | 2000 Sep

REPOSITORIES: biostudies-literature