Title: Designing Primers
Course: BTEC-1100-301-302-303-F21 > Section: Module 3 - Polymerase Chain Reaction

Every PCR requires two short DNA molecules, called primers, that are specific to the gene you are trying to amplify. The sequence of these primers is determined by the sequence of the DNA that you are trying to amplify.  In the previous section we were able to find our gene in the NCBI database and we found the sequence of that gene.

DNA sequences in the NCBI database are always published from the 5’ to 3’ direction, but remember DNA is composed of a coding strand and a complementary template strand running 3’ to 5’. Two primers are designed for the PCR; a forward primer that extends one strand from the start codon to the stop codon and a reverse primer that extends from the stop codon to the start codon. That looks something like this:

A tutorial on how to design primers is found here: PCR Primer Design

There is also a video tutorial on the next page.

There are some general concepts to use when designing primers:

1. The 3’ end of any primer, forward or reverse, should be complementary to the template DNA. Most primers should be around 15-30 bases in length to give a melting temperature of approximately 55-60˚C.  Additional sequences can be added at the 5’ ends of primers.  These sequences are not complementary to the template DNA and may contain any desired genetic information. We will be adding the LIC cloning sites to our primers so that we may clone our gene of interest into a plasmid.
2. Multiple Gs and/or Cs at the 3’end should be avoided if possible
3. Forward and reverse primers should not have complementary 3’ ends which can cause the formation of primer-dimers.
4. Each primer should have about 40-60% of Gs and Cs.

Remember: The next step is to come into the lab to practice with the materials, view a demonstration, or to even just ask questions. Also, if you are ready to take the lab final, please inform your instructor. The lab final will be performed by yourself and will be graded with a rubric.