PCR Primer Design Guidelines

Hopkins Lab

Version 1.4

PCR Primer Design Basic Guidelines Ideally primers targeting mRNA should bind separate exons. Primers usually between 18-25 bases long. Primer GC content ~50% • • •

Amplicon should not have G/C content in excess of 60% (Ideally 4060%). High GC content may not denature well during cycling and also susceptible to non-specific interactions Balanced distribution of G/C and A/T bases.

3’ terminal position needs to be a G or C, but no more than 2 G or C in last 5 bases is best. Avoid 3’ terminal T. No runs of single bases greater then three bases especially for G or C. Tm of primer 55-65°C and pairs should not differ by more than 1-2°C. Match primers for ΔG more than Tm. ΔG no more than -10 Kcal/mol. Avoid secondary structure or sequence complementary at 3’ ends (as this leads to the formation of primer dimers, which depletes the effective primer concentration, compromises efficiency). PCR Primer Design when using Topoisomerase cloning kits (e.g. StrataClone™ PCR Cloning Kit)

• Avoid including the sequences C/TCCTT or AAGGG/A in the PCR primers. The presence of one of these sequences in the primer creates a topoisomerase I-binding site (CCCTT, or TCCTT) in the PCR product. • Avoid initiating a PCR primer with a 5´-T residue. When T is present at the 5´end of the primer, the resulting PCR product contains a template-directed 3´-A residue. Non-template A-addition by Taq DNA polymerase is least efficient adjacent to a 3´-A residue and is most efficient adjacent to a 3´-C residue. • Do not phosphorylate the 5´-ends of PCR primers. Topoisomerase I strictly requires a 5´-hydroxyl group as a substrate for the DNA strand-joining reaction.

Real Time primer design specific guidelines (SYBR Green I) Amplicon length usually 100-150 bp if using SYBR green can be up to 250 bp. Use melting curve analysis to identify primers that dimerise. Page 1 of 3

Hopkins Lab

Version 1.4

Avoid GC rich amplicons if possible (difficult to melt). Can check amplicon with Mfold program.

Primer Design Software. There are many programs for primer design, commercial and free the following work fine but so do many other programs. You don’t have to use these if you want to use other programs.

Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi [Also available through Jemboss package under nucleic, primers, eprimer3 menu.] Product Size Ranges: Enter the size range of the Amplicon you wish Number To Return: number of primer alternative usually set to 5 or 10 Max Tm Difference: difference in primer Tm set to 2 Primer GC% Min: set to 40 Opt: set to 50 Max: set to 60 Max Self Complementarity: set to 2 to start with may need to relax this if no primers found i.e. increase value by 1* Max 3' Self Complementarity: set to 0 to start with may need to relax this if no primers found i.e. increase value by 1* Max Poly-X: set to 3 *lower values more stringent.

These are basic guide to primer3 but provide a good starting point for primer design but these values are not always ideal for every primer you need to design just most of them. Reading the accompanying help pages etc. on the web site will give you a more in depth guide to the options.

NetPrimer http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html This software can be used to check your primers from primer3 and any optimisation/changes you do on your primers. Remember primer3 is just a starting point in the design of primers it is very rarely the endpoint you need to check the primers fully before ordering. You can also just design primers by hand without primer3 and check them with Netprimer for secondary structure, dimmers etc. Its not compulsory to use primer3, you can do it yourself! Read the online help guide it is short. Quick guide The higher the primer rating the better 100 is best. Tm and % GC should be similar between forward and reverse primers but more crucial to get similar ΔG, 3’ end stability, ΔH, ΔS and 5’ end ΔG. No intra-primer homology beyond 3’ base pairs Page 2 of 3

Hopkins Lab

Version 1.4

Avoid secondary structure or sequence complementarity (>2-3 bp) especially at 3’end. While avoiding hairpin loops, dimers and Cross dimers, palindromes and repeats and runs. Though you may have to live with some of these.

BEFORE ORDERING PRIMERS After performing the checks with Netprimer you should check your primers with the BLAST program to ensure that they are specific for the gene you designed them for. http://www.ncbi.nlm.nih.gov/BLAST/ Select the Basic BLAST program option, choose nucleotide blast. • • •

Choose database others nucleotide collection Under program selection choose somewhat similar sequences Check Algorithm parameters are same as below.

If all is fine go ahead and order. Good Luck. IF UNSURE ASK. Page 3 of 3