Words similar to overlap-extension

Example sentences for: overlap-extension

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Oligonucleotide primers for the PCR reaction were based on the published sequence of murine Rab24 [ 24 ] . Kpn1 and BamH1 restriction sites were added to the 5' and 3' primers respectively, to facilitate cloning of the PCR product in frame with a 5' sequence cassette encoding a myc epitope (EQKLISEEDL), previously introduced into the pCMV5 expression vector [ 78 ] . The Rab24 cDNA was altered by overlap-extension PCR [ 79 ] , using appropriate mutator oligonucleotides and Pfu DNA polymerase (Stratagene Inc., La Jolla, CA), so as to introduce amino acid substitutions at D123 (D123I), T120 (T120A), S22 (S22N), S67 (S67Q), and a deletion of residues 127 through 130 (ΔEDRR).
pCMV5 expression vectors containing Rab24 and Rab1B were engineered by overlap-extension PCR to encode the following chimeric proteins: Rab24/Rab1B, consisting of residues 1-165 of Rab24 [ 24 ] fused to residues 163-201 from Rab1B [ 80 ] ; Rab24(D123I)/Rab1B, consisting of residues 1-165 of Rab24 with the D123I substitution, fused to residues 163-201 of Rab1B; Rab1B/Rab24, consisting of residues 1-162 of Rab1B fused to residues 166-203 of Rab24; and Rab1B(N121I)/Rab24, consisting of residues 1-162 of Rab1B with the N121I substitution, fused to residues 166-203 of Rab24.