Table 1.

Templates and primers used in the PCR walking technique to clone and sequence arfI

PCRTemplatePCR primers
NameaSequenceb
1 C. xylanolyticagenomicF15′-GAR GCN GCN CAR TGG GT-3′ (forward)
DNAR15′-GCR TTR TTR TTR AAD ATR TT-3′ (reverse)
2pUC19-EcoRI libraryc F25′-CAG TCA CGA CGT TGT AAA ACG ACG GC-3′ (forward)
R2d 5′-CCA AGT TTG ATG CCA CGA CTG TTC-3′ (reverse)
3pUC19-KpnI libraryc F25′-CAG TCA CGA CGT TGT AAA ACG ACG GC-3′ (forward)
R35′-GAT ATC CCA GGG TTT ATC ACG ACC G-3′ (reverse)
4pUC19-HindIII libraryc F45′-CGC AGC ATC ACA AGT TCC TCA TGC-3′ (forward)
R45′-TCA CAC AGG AAA CAG CTA TGA CCA TG-3′ (reverse)
5 C. xylanolyticagenomicF55′-GGT CGT TGG TGA AAT ACA GCG G-3′ (forward)
DNAR55′-GAC AAA TCG CTC CCA CCG AAC AC-3′ (reverse)
  • a The priming sites of F2 and R4 complemented regions within the multiple cloning site of pUC19.

  • b A, adenosine; T, thymidine; G, guanosine; C, cytosine; R, adenosine or guanosine; N, adenosine, thymidine, cytosine, or guanosine; D, adenosine, thymidine, or guanosine.

  • c Library of C. xylanolytica genomic DNA created by restriction with an enzyme and ligated into pUC19.

  • d The second and fourth nucleotides of primer R2 (cytosine and adenosine, respectively) were identified incorrectly in the amplicon from PCR 1 and did not correspond to the homologous nucleotides in amplicons from PCR 4 and 5. However, their distances from the 3′ end did not compromise the efficacy of R2 as a primer for PCR 2.