The use of a PCR cloning and screening strategy to identify lambda clones containing the hupB gene of Anabaena sp strain PCC7120

Two lambda clones containing the hupB gene of Anabaena sp. strain PCC7120 were isolated from a genomic DNA library using a cloning and screening strategy which involved the polymerase chain reaction (PCR). Two highly conserved stretches of amino acids were identified within the HupB sequences of various organisms and two degenerate PCR primers were synthesized based on these sequences. Upon amplification of the Anabaena genomic DNA using these primers, a 278-bp DNA fragment was obtained. By sequencing this PCR fragment it was established that this was a part of the Anabaena hupB gene. Based on this sequence, two high-stringency PCR primers were synthesized that were used to isolate two lambda clones containing the hupB gene by PCR-screening of an Anabaena genomic library. By using a modified screening procedure the two lambda clones were directly isolated from those plates which appeared positive in PCR mass-screening and in which the plaques were cross-contaminated during the mass screening.