u00b0 c for 30 seconds and annealing and extension at 70 u00b0 c for 3 minutes and 25 cycles of denaturation at 94 u00b0 c for 30 seconds and annealing and extension at 68 u00b0 c for 3 minutes amplified products were used as templates in a nested pcr reaction using the previously described cycling parameters the reamplified products were then analyzed by southern blotting using oligonucleotide probes specific for p110 u03b4 probes 100 ng each were end labelled with 32 p u03b3atp and hybridized and washed under standard conditions frisch and sambrook the sequences of the two probes were gatgcggaacggctgctccaggg seq id no 11 and ccagggaccacagggacacagag seq id no 12 the specific 5 39 race pcr products identified in this manner were gel purified and subcloned into the ta vector pcrii invitrogen san diego calif according to the manufacturer 39 s instructions three independent clones were sequenced to ensure the veracity of the 5 39 sequence a full length cdna for p110 u03b4 was assembled from clones 249 m 928 and the 5 39 race pcr products the 5 39 race product was used as a template in pcr using the 5 39 primer agttacggatccggcaccatg gactacaaggacgacgatgacaag ccccctggggtgga ctgccc seq id no 13 and the 3 39 primer ccacatgtagaggcagcgttcc seq id no 14 the 5 39 primer includes a bam hi site underlined and sequences that encode the flag peptide sequence dykddddk seq id no 15 shown in parenthesis which is recognized by the m2 anti flag monoclonal antibody kodak scientific imaging systems new haven conn the resulting pcr product was digested with bam hi and afl ii and was ligated along with an afl ii pvu i fragment derived from the clone m 928 and a pvu.