A primer constructed on this sequence motif (primer P17, Figure 2) has enabled us to restrict the amplification of FMO homologs to sequences belonging to this sequence cluster.
The PCR reaction solution (50 μl) for preamplification contained 10 μl of the digestion mix, 75 ng of each primer constructed using the adaptors, 0.2 mM dNTP mix, 1.5 mM Mg2+ and 1 U of Taq polymerase (Invitrogen).
Three cytochrome P450 monooxygenase (CYP) genes, designated pb-1, pb-2 and pb-3, were isolated from the white-rot fungus, Phlebia brevispora, using reverse transcription PCR with degenerate primers constructed based on the consensus amino acid sequence of eukaryotic CYPs in the O2-binding, meander and heme-binding regions.
Regions of the recA gene, varying in size between 613 and 677 nucleotides, were sequenced for Propionibacterium acidipropionici, P. cyclohexanicum, P. freudenreichii, P. jensenii, P. microaerophilum and P. thoenii using degenerate consensus primers constructed by aligning recA sequences of some actinobacteria.
Clones were sequenced using T7 and Sp6 primers as well as custom primers constructed from the internal sequence of the cDNA clone.
Gaps in the sequence were filled by direct PCR sequencing, using primers constructed to anneal to each end of neighboring contigs.
For low-quality DNA, extracted from faeces or museums material, two to seven overlapping PCR products were amplified with primers constructed on the basis of sequences from conspecifics (respective primers are available from the authors upon request).
A ChIP assay was performed using RA-FLS over-expressing LIP and primers constructed from the human RANKL promoter sequence, which amplify sites including the C/EBPβ consensus site-4.
Note however that relaxation of selective constraints in pseudogenes may result in elevated substitution and indel rates, resulting in violation of the assumption of non-biased targeting of the primers constructed from an alignment of apparently functional gene sequences.
Five primers were constructed; two outer primers F3 and B3, two inner primers: forward inner primer (FIP) backward inner primer (BIP) and loop forward primer (LF).
In step 3, the whole primer constructs were tested for potential dimer formation.
