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Figure 3 | BMC Molecular Biology

Figure 3

From: Selection strategy and the design of hybrid oligonucleotide primers for RACE-PCR: cloning a family of toxin-like sequences from Agelena orientalis

Figure 3

The comparison of CODEHOP and PaBaLiS primers. (A) Schematic representation of the CODEHOP primer, based on the original design by Rose et al. [31]. The maximum degeneracy is aimed at the 3' region, which reduces the amplification efficiency (due to competition by "wrong" 3' end sequences for annealing, these will disallow amplification) and very high probability of dimer formation by such oligos (e.g. fully degenerate 3-4-5 or 6-mers will all able to dimerise). (B) Schematic representation of the PaBaLiS primer design. Note that maximum degeneracy is achieved in the middle of the primer, minimising the number of possible mismatches in the middle of the primer and increasing the stability of the hybrid (primer/cDNA). Low degeneracy (and high sequence conservation) at the 3' end ensured the highest specificity of annealing, amplification and allows to easily avoid primer dimers. (C) The actual distribution of degenerate bases for PaBaLiS primers designed in this study. The choice of oligonucleotides for this analysis includes the 22 oligonucleotides of identical length (20-mer long oligonucleotides, based on 7 amino acid long stretches of partially of fully homologous protein sequences) of the 44 oligonucleotides designed in total. The horizontal axis indicated the nucleotide degeneracy in triplets approximately corresponding to each of the 7 amino acid positions used to generate these 20-mer oligonucleotides. The mean degeneracy calculated as total degeneracy at this position (due to both the amino acid and the genetic code degeneracy) divided by the number of oligonucleotides). The numbers of the bars indicate actual values. Note that decreased overall degeneracy at 5' end is due to the use of "best guess" based and triplets, but not because amino acid degeneracy was lower at this position. (D) The actual distribution of inosine substitutions in the same PaBaLiS primers as in (C). The use of inosines allowed to decrease overall degeneracy without introducing mismatches in the middle of the primers,

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