Designing Primers

• Primer


  Short nucleic acid sequence that provides a basis for DNA synthesis or DNA sequence to be amplified
• Probe


  Longer nucleic acid sequence, ranging from 100 to 1000 or sometimes 10,000 nucleotides in length, that functions to detect the presence or absence of target nucleic acid present in a sample
• Primers


  • Designed in pairs (forward and reverse)
  • Binds from where the amplification will start
  • No radio-labelled and safe to use
• Probes


  • Generally labelled with radiolabeled or non-labelled compounds
  • Function is to detect the presence or absence of target nucleic acid present in a sample
  • Bind with a target location
• Primers and probes both bind or anneal to the DNA template and act as starting points for DNA polymerase
• Amplification


  Method for synthesis of DNA done through the Polymerase Chain Reaction (PCR) and done in a thermocycler
• BLAST


  Basic Local Alignment Search Tool
• Base pair


  Used to measure the size of an individual gene within a DNA molecule. The total number of base pairs is equal to the number of nucleotides in one of the strands.
• DNA Polymerase


  The enzyme used for producing DNA molecules, however during PCR, it can only initiate DNA synthesis with the presence of primers
• Important considerations when designing primers

  • What species do you intend to analyze? (Human, plant or animal)
  • Are there previously published primer sequences available? If YES, can you use it?
  • If NO, then select your gene of interest (GOI)
  • PCR primer design is sometimes a matter of trial and error. It is important that the designer has knowledge on the best software to assist on designing.
• Good primer characteristics


  • Tm or melting temperature in the range of 52 – 65 Celsius
  • Absence of dimerization capability
  • Absence of hairpin formation
  • Lack of secondary priming sites
  • Low specificity binding at the 3' end
  • GC content of 40 to 60%
• Selecting a Gene of Interest (GOI)


  1. Access the NCBI Map Viewer from the NCBI's home page

  2. Click the upper left ALL Database and choose 'nucleotide'

  3. Type your GOI in the search engine
• Gene of Interest


  • Colibactin B (clbB)
  • clbB Escherichia coli
• 8 Basic Concepts for Designing Primers for a Standard PCR

  • Short or long enough: Primer length
  • Temperature is important: Tm and Ta
  • High or low bondage: GC content (Primers with 40% to 60% GC content)
  • Ends matter: 3' and 5' end of the primer
  • Repeat and secondary formation of primer (or e.g. ATATATATA it can result in mispriming)
  • Product length
  • Cross binding: check for homology
  • Computer programs that make it easier
• Escherichia coli strain 240418:M-17-Peretz putative hybrid polyketide-non-ribosomal peptide synthetase (509 bp)
• Browse down the Primer BLAST (uncheck the specificity check)
• clbB ds DNA cgtatta cccggcccac attcgataaa cagcgtgggt tgtaccaaca gttgcgccgc accttcgcta aacagcac
• in silico Testing NetPrimer http://www.premierbiosoft.com/crm/jsp/com/pbi/crm/clientside/EligibleForDiscountLoginForm.jsp?LoginForFreeTool=true&PID=3
• Oligo Calc: Oligonucleotide Properties Calculator http://biotools.nubic.northwestern.edu/OligoCalc.html
• The ratings must range between 80-100; no hairpins; no repeats; and if possible no self dimers.