Primers applied in analysing of gut content of predators should ideally target short sequences of multiple-copy DNA because of the degraded nature of the prey derived sequences [13 (link),16 (link),17 ,36 (link)]. The ribosomal DNA is therefore often used as a target for PCR amplification in diet studies because ribosomal DNA (rDNA) genes are repeated tandemly in high copy numbers and are highly conserved within species [37 (link)]. We designed 'universal' PCR primers that amplify a short, but fairly variable region of the 28S rDNA from all eukaryotes tested. We also designed three blocking primers intended to bind to the Euphausia superba sequence amplified by the universal primers. The primers used in this study are given in Table 1 and shown aligned with the krill sequence in Figure 2.
The blocking primer, 'Short28SR-blkKrill3'c3' overlapped with the 3' end of the reverse universal primer, but extended into krill-specific sequence and was modified with a C3 spacer at the 3'-end (Figure 2). We needed a modification which was 100% synthesized (i.e. no oligos missing it) and which was stable (i.e. no degradation or enzymatic removing of the modification after synthesis). Even if just a small percentage of the blocking primers were to prime amplification of predator DNA as a result of not having the 3' modification, this would render the procedure unworkable. C3 spacer (3 hydrocarbons) CPG is a standard primer modification available from most suppliers of custom oligonucleotides. Adding this modification to the 3'-end of an oligonucleotide prevents elongation during a PCR without noticeably influencing its annealing properties. Because oligos are synthesized from a 3' to 5' direction, all molecules will be modified with the 3' modification. Modifying the 3'- end with a phosphate group (as chosen by Liles et al. [35 (link)]), a phosphate ester, or using an inverted 3'-3' linkage would also prevent elongation. However, side reactions during deprotection of the oligonucleotide or enzymatic impurities may free the 3'-hydroxyl group to a small extent, and these methods are not so effective in blocking as C3 spacers CPG [38 (link),39 (link)].
Because finding an appropriate binding site for a species specific primer next to a binding site of a universal primer is often difficult, a krill specific blocking primer, Short28SF-DPO-blkKrill overlapping with the 3'end of the forward universal primer and having an internal modification of five deoxyinosine (dI) molecules in addition to the C3 spacer modification was also designed (Figure 2). Very long conventional oligonucleotides often do not work. In general, primers longer than 25 bases are rarely used since their Tms can be over 70°C, which is too high for effective PCR cycling [40 (link)]. Long primers also often generate many non-specific bands resulting from non-specific annealing. A dual priming oligonucleotide (DPO) [41 (link)] contains two separate priming regions joined by a polydeoxyinosine linker. DPOs does not suffer from the limitations of a high Tm since the linker assumes a bubble-like structure resulting in two primer segments with distinct annealing properties. Furthermore, the bubble-like structure of linker efficiently prevents primer-dimer and hairpin structure formation [41 (link)].
The Short28SR-blkKrill3'c3 and the Short28SF-DPO-blkKrill blocking primers were both designed to prevent annealing of the unmodified version of the universal primer on krill sequences. Further a third krill specific blocking primer situated between the two universal primers was tested (Figure 2). This was an "elongation arrest" primer ([42 (link)]; Figure 1) and also had a C3 spacer at its 3' end.
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