How to Design Primers

For PCR using Primer3 website

Yousef Alhashem
2011
PCR
• Polymerase Chain Reaction
• in vitro amplification of DNA using heat
stable DNA polymerase enzyme. DNA
• Three cycling steps:
– Denaturation
– Annealing
– Elongation
Denaturation
• At the end of each cycle the amount of
DNA is doubled.
• Reagents needed:
– Template (DNA)
– Polymerase (e.g. Taq polymerase)
Annealing
– Primers
– Nucleotides (A,T,C,G)
– Magnesium, potassium, and buffer

Elongation
Primers

• Short sequence of nucleotides

– ~20 nucleotides
• Required to initiate DNA synthesis by DNA
polymerase.

• Good primers:
– About 20 nucleotides long
Denaturation
– 50-60% are G+C
– Have melting temperature ~60
– No more than 3 G or C at the 3’ end
– Not self complementary

Annealing

Elongation
Primers Design

• Use Primer3 website to design primers for known regions of DNA

• http://frodo.wi.mit.edu/primer3/
• Primer3 developed by Steve Rozen and Helen Skaletsky.
• Primer3-web is maintained by Steve Rozen.
Obtain DNA

• Find the DNA sequence of the region you want to amplify

• You can use the NCBI website for that.
• http://www.ncbi.nlm.nih.gov/
• Select Gene database and search for you gene of interest
Obtain DNA

• If you search for klf1 for example, you will get klf1 genes in several species
• Select the correct species. I select mouse (Mus musculus) klf1.
Obtain DNA

• If you search for klf1 for example, you will get klf1 genes in several species
• Select the correct species. I select mouse (Mus musculus) klf1.
Obtain DNA

• Go down the page until you reach genomic section

• Select GeneBank if you want to learn more about the structure of your
gene.
• Select FASTA to get only DNA sequence of the gene

Go down
Obtain DNA

• Copy the region of DNA you want to amplify.

• Go to Primer3 website, http://frodo.wi.mit.edu/primer3/
Primer3

• Paste the DNA sequence into the provided space

• Tell the website where do you want you primers to be by adding “[“ and
“]” around the sequence of interst.
• Tell the website what is the size of amplicon you would like to see.
• Hit “Pick Primers”
Primer3

• You will get the best pair of primers at the top of Primer3 Output screen.
• The primers’ sequences , the melting temperature as well as other
information will be shown.
• You need to make sure your primers are specific using NCBI blast engine.
• http://www.ncbi.nlm.nih.gov/tools/primer-blast/
T.h.a.n.k
Y.o.u