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 design

Terminal Nucleotides: 3 terminal positions are essential for controlling mispriming 3 end of primers must be carefully selected to prevent homologies within the primer pair known as primerdimer where the PCR product is amplification of primers GC content and Tm Primers with 50% G+C content have a Tm between 5662oC Tm and GC content should be similar between primer pairs
A/T = 2C G/C= 4C

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 Isolate 2 Isolate 3 Isolate 4 5 ATGGCATCTGACTGACACCACCTCAATCAA 3 5 ATGCCATCTGACTGACACCACCTCAATCAA 3 5 ATGGCATCTGACTGACACCACCTCAATCAA 3 5 ATGGCATCTGACTGACACCACCTCAATCAA 3

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

Inosine
Isolate 1 Isolate 2 Isolate 3 Isolate 4 Primer sequence 5 ATGGCATCTGACTGACACCACCTCAATCAA 3 5 ATGGCATCAGACTGACACCACCTCAATCAA 3 5 ATGGCATCTGACTGACACCACCTCAATCAA 3 5 ATGGCATCCGACTGACACCACCTCAATCAA 3 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 primer 3 TACGGCGTTAAGCAATAATGAAGCTAGGC 5 5-3 primer sequences F - atgcc R - 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