Concepts of Primer Design

• Design is crucial to successful amplification of target DNA by PCR

• Determine the size and location of PCR product• Well-designed primers can deter amplification of

background and non-specific products• Poorly designed primers result in no or very low

PCR product yield• Several computer programs are available for

selecting primers

Concepts of primer design

Goal Balance specificity and efficiency of amplification

Primer selection/analysis software assess these two criteria by evaluating the following criteria:– Primer length– Terminal nucleotide– G + C content and Tm– PCR product length– Placement in target

sequence

Concepts of primer design

• Primer Length:

– Primers between 18-24 nucleotides in length tend to be very sequence-specific if the annealing temperature is set within a few degrees of the primer Tm

– Optimize PCR by using the minimum primer length that ensures Tm of 54oC or higher

Concepts of primer designTerminal Nucleotides:

– 3’ terminal positions are essential for controlling mis-priming

– 3’ end of primers must be carefully selected to prevent homologies within the primer pair known as primer-dimer where the PCR product is amplification of primers

GC content and Tm– Primers with 50% G+C content have a Tm between 56-

62oC– Tm and GC content should be similar between primer

pairs– A/T = 2°C– G/C= 4°C

Concepts of primer design

• Use multiple sequences to design/validate primers if possible– WHY?

• PCR Product Length and Placement within Target Sequence– Length of PCR product affects efficiency of

amplification– Size of the PCR product depends on application

Degenerate primers• Primers with mixed base pairs Isolate 1 5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’Isolate 2 5’ ATGCCATCTGACTGACACCACCTCAATCAA 3’Isolate 3 5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’Isolate 4 5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’

Primer sequence 5’ ATG(G/C)CATCTGACTGACACC 3’50% of primer synthesized with each nucleotide

• InosineIsolate 1 5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’Isolate 2 5’ ATGGCATCAGACTGACACCACCTCAATCAA 3’Isolate 3 5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’Isolate 4 5’ ATGGCATCCGACTGACACCACCTCAATCAA 3’

Primer sequence 5’ ATGGCATC(I)GACTGACACC 3’

Concepts of primer design

• Hypothetical DNA and primer sequences

5’ ATGCCGCAATTCGTTATTACTTCGATCCG 3’*reverse primer 3’… TAGGC 5’

5’ ATGCC … 3’ *forward primer3’ TACGGCGTTAAGCAATAATGAAGCTAGGC 5’

5’-3’ primer sequencesF - atgccR - cggat

Primer design exercise• You will be provided a hand-out with the DNA

sequence for Salmonella enteritidis invA

• Design a set of primers to amplify a 600 base pair region of this gene

• Write down the sequence of both your primers in the 5’ to 3’ orientation (requested by primer synthesis companies)

• Calculate G+C content for your primers to make sure they are similar