Primer Design Worksheet

The specificity of PCR depends on the primers used to amplify a given segment of DNA. These primers are on opposite sides, and strands, or the target fragment. Good primers are specific to a single DNA sequence. For eDNA this is particularly important as our DNA extraction contains many microbial genomes from an environmental sample. In this lab, we want to only amplify a short DNA sequence from the spotted salamander: Ambystoma maculatum.

We will be designing primers based on the A. maculatum cytochrome b gene.

You can view the record for this accession here: http://www.ncbi.nlm.nih.gov/nucleotide/118202038

· How long is this sequence? How many “bp?”

· Note that this gene is found outside the nucleus. What organelle has its own DNA?

· When was the sequence published? What was the title of the paper?

· What is the string of letters following “translation=” represent?

The Pubmed accession number is: EF036637.1

Copy and paste this sequence into the “Enter accession” box at the top of the primer design site:

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

Under “Database” choose “nr.” This is all published nucleotide data.

Under “Primer Parameters” change the PCR product size “max” from “1000” to “100”

Under “Organism” we will add organisms from the aquatic environment of out spotted salamanders. First click “Add more organisms” then add:

Ambystoma maculatum (taxid:43114)

Branchinecta lynchi (taxid:577405) (this is the “fairy shrimp” that is often found in the same vernal pools)

Lithobates sylvaticus (taxid:45438)

Fairy shrimp

Under “Primer specific stringency” change the “total mismatches from 2 to 3.

We will leave everything else as a default.

Click:

You will be prompted with the following.

Select both of these Cyt B submissions and hit “Submit”

Your results should look like this:

The top “Template” bar is a graphical representation of your submitted sequence. The program has returned 10 primer pairs.

Scroll through the Detailed Report.” Note that none of the entries bind to published fairy shrimp or wood frog sequences.

Each report contains the following:

Note the forward and reverse primer sequences bind to the two different strands (plus and minus) and in this example each has a length of 22bp.

The distance between the beginning of the forward primer and beginning of the reverse primer is the product length (465-395 = 71bp).

The “Tm” s the predicted annealing temperature of this primer to the target DNA sequence.

The GC% is the percentage of guinines and cytosines. We typically like something around 50%.

The “self complimentarity” score is a measure of how likely the primer is to fold on itself. We like to see values below 6.00.