Table 1 Algorithm parameters and default values for primer/probe design
From: barCoder: a tool to generate unique, orthogonal genetic tags for qPCR detection
Purpose | Description of constraint or check | Default parameter valuesa | ||
|---|---|---|---|---|
Forward primer | Reverse primer | Probe | ||
Primer/probe Constraints | Minimum length (bases) | 20 | 20 | 20 |
Maximum length (bases) | 24 | 24 | 30 | |
Minimum Tmb (°C) | 58 | 58 | 68 | |
Maximum Tmb (°C) | 60 | 60 | 70 | |
Minimum G+C Content (%) | 40 | 40 | 40 | |
Maximum G+C Content (%) | 60 | 60 | 60 | |
Number of C’s ≥ G’s in probe | n/a | n/a | True | |
Primer/probe Checks | Maximum G Homopolymer Length (bases) | 3 | 3 | 3 |
Maximum A/T/C Homopolymer Length (bases) | 4 | 4 | 4 | |
No G on 5ʹ end of probe | n/a | n/a | True | |
2 G/C’s in last 5 bases of 3ʹ end of primers (GC clamp) | True | True | n/a | |
No start codons present | True | True | True | |
Primer/probe Stem-loop Checks | Minimum stem-loop hydrogen bonds | 14 | 14 | 14 |
Minimum stem-loop palindrome length (bases) | 5 | 5 | 5 | |
Maximum stem-loop palindrome length (bases) | 100 | 100 | 100 | |
Maximum stem-loop gap size (bases) | 11 | 11 | 11 | |
Maximum stem-loop mismatches | 1 | 1 | 1 | |
BLAST checks | Thresholdc to reject primer/probe based on BLAST similarity | 0.85 | 0.85 | 0.85 |