ProbeMelt

DECIPHER contains multiple functions for predicting the effectiveness of oligonucleotides, as described in:

• FISH probes (CalculateEfficiencyFISH):

  ES Wright et al. (2014) "Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification." Applied and Environmental Microbiology, doi:10.1128/AEM.01685-14.

• Primers (CalculateEfficiencyPCR):

  ES Wright et al. (2014) "Exploiting Extension Bias in PCR to Improve Primer Specificity in Ensembles of Nearly Identical DNA Templates." Environmental Microbiology, doi:10.1111/1462-2920.12259.

For an in-depth tutorial on oligonucleotide design, see the associated vignettes on the Documentation page.

How do I calculate hybridization efficiency?

First it is necessary to install DECIPHER and load the library in R. Next, specify the oligonucleotide probe or primers and their target sequences:

Hide output

library(DECIPHER)
> 
> oligos <- c("TCACCTGGCCATGTCGC", "TCACCTGATCATGTCGC")
> # reverse complement of first oligo:
> targets <- rep("GCGACATGGCCAGGTGA", 2)
> 
> CalculateEfficiencyFISH(oligos, targets,
+ temp=46, # hybridization temperature
+ P=250e-9, # probe concentration (molar)
+ ions=1, # molar Na+ concentration
+ FA=35) # formamide concentration (% v/v)
      HybEff       FAm     ddG1       dG1
1 0.56553679 37.568501 0.000000 -12.08644
2 0.04949342  6.211841 4.078027 -10.04512
> 
> CalculateEfficiencyPCR(oligos, targets,
+ temp=64, # annealing temperature (Celsius)
+ P=4e-7, # primer concentration (molar)
+ ions=0.225, # Na+ equivalent concentration (molar)
+ taqEfficiency=FALSE) # TRUE if using Taq polymerase
[1] 0.8868337419 0.0005365686