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  The features of a good qPCR primer pair

What attributes make a good primer pair?

There is such a thing as a good and bad primer. The former are the perfect candidates used to amplify the region you are interested in with 100% efficiency, without amplifying anything else in the genome. The latter, however, are those which completely destroy qPCR results. They are inefficient, have low specificity and cost you valuable time through further optimizations.

In the video below, I will go through the attributes of what makes a good primer pair.

A few notes about probe attributes

Probe-based assays also require an additional primer, known as a probe. For these, there are a few additional recommended attributes:

  • Binding location: Design the probe to bind near, but not overlapping, the forward or reverse primer.
  • Melting temperature: The melting temperature of the probe should be around 6 - 8 °C higher than that of the forward and reverse primers.

Summary

Here are the important points to summarize this lecture:

  • Keep the PCR product size relatively small (70 - 200 bp).
  • Primer lengths should be ~20 bp long each.
  • The melting temperature of each primer should be between 59 - 65 °C. The primers in the primer pair should have identical melting temperatures (within 1 °C of each other).
  • The GC content for each primer should be between 50 - 60%. The primers in the primer pair should have identical GC content.
  • Ideally, primers should contain a GC clamp. This ensures the primer will completely bind on the template.
  • Avoid primers with repeat nucleotide sequences, e.g. TTTTT.
  • Separate the forward and reverse primer by an intron. This limits the chance of genomic DNA amplification.
  • If using Oligo(dT)s during reverse transcription, then design the primer pairs to bind near the 3' of the gene.
  • If using random hexamers during reverse transcription, then design the primer pairs to bind near the 5' of the gene.
  • Primer pairs should be specific to the target of interest. No other unintended targets should be identified during the BLAST algorithm.