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

Stability

• should not form hairpins

• should not form homodimers

• should not form heterodimers

• should be stable in the 3’ end

  • so the polymerase can extend

Specificity

• 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

Evaluation

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

\(TP\)

True Positive number

\(TN\)

True Negative number

\(FP\)

False Positive number

\(FN\)

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”

Tools

• Primer 3
• Primer BLAST