Introduction

Juniper is a tool for the design of oligonucleotides in Pearl & Prodromou's Recursive PCR process (Protein Engineering, 5, 827-829 (1992)). This is a PCR technique for gene synthesis which requires niether phosphorylation nor ligation, gives high yields, and has the potential for the total synthesis of much larger genes than other established techniques. Whilst recursive PCR is very powerful, the design of oligonucleotide sequences by hand can be time-consuming.

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Juniper consults codon usage tables to ensure good expression, inserts or removes restriction sites where appropriate and checks for sequence overlap or complmentarity to avoid non-specific priming. It then designs overlapping regions with melting temperatures in a specified range to yield an even number of oligonucleotides of similar length suitable for Recursive PCR.

Methodology

1. Given the desired protein sequence, Juniper consults codon usage tables to employ the most efficient codon for each residue. Alternative codons and their efficiencies are recorded for each residue.
2. A table of restriction enzymes is used in combination with the alternative codon choices to calculate all possible restriction sites involving each residue.
3. Existing restriction sites are flagged. Sites in the list of banned enzymes that are present for the selected highest-usage codons are removed, providing that the changes meet codon usage thresholds.
4. Sites which occur twice in the sequence are removed, providing that the changes meet codon usage thresholds.
5. For each permitted enzyme that currently does not have a restriction site within the sequence, all possible sites are considered. If the best of these sites meets codon usage thresholds and exists within a sufficiently large gap, it will be added to the sequence.
6. Regions of sequence identity and complementarity with each strand are located. If a region exceeds a threshold length, changes that will eliminate overlap are considered. If the best of these meets codon usage thresholds, it will be applied.
7. The desired oligo length is used to calculate an actual oligo length, given the length of the sequence and the necessity for an even number of oligos. If the actual length does not deviate from the desired length by more than the oligo length slop, overlap sites are located.
8. Each overlap site is extended until the annealing temperature matches the desired range. If this is not possible, or if the extension takes the oligo length outside the permitted range, the process is abandoned.
9. The oligo designs are presented to the user.