Class 15: Designing primers


Andrés Aravena

December 02, 2021

Conditions for primers

What is a good primer

  • It is a short sequence, 16-24bp

  • It binds spontaneously to the target

  • It does not bind easily to other things

  • It works well with the other primer

  • It works well with the polymerase

In other words

  • It has to be thermodynamically stable

  • It has to be taxonomically specific

These two conditions imply that the sequence must be short, but not too short


  • should not form hairpins

  • should not form homodimers

  • should not form heterodimers

  • should be stable in the 3’ end

    • so the polymerase can extend


  • should match the target organism only once
    • even if there are some mismatches
  • should not match other organisms
    • even if there are some mismatches

Four possible outcomes

When we use a primer to detect a target, four things can happen

  • The primer binds to the target

  • The primer binds to something else

  • The primer does not bind to the target, even if the target is there

  • The primer does not bind to anything, and the target was not there

These cases are called: True Positive, False Positive, False Negative, and True Negative


When we test a possible primer, the four outcomes may happen

We measure the number of each case, using a database of all possible sequences

True Positive number
True Negative number
False Positive number
False negative number

Sensitivity and Specificity

\[\begin{aligned} \text{Sensitivity}&=\frac{TP}{TP+FN}=\frac{\text{Detected}}{\text{All targets}}\\ \text{Specificity}&=\frac{FP}{TN+FP}=\frac{\text{Not targets}}{\text{Not detected}} \end{aligned}\]

“Say the truth, all the truth, nothing but the truth”