1. IN THE NAME OF GOD

2. PCR Primer Design Lecturer: Dr. Farkhondeh Poursina

3. Definition PCR primer design is the creation of short nucleotide sequences for use in amplifying a specific region of DNA.

4. Polymerase chain reaction (PCR)  Developed in 1985 by Kary Mullis  Amplifies a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA Sequence.  PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.

5. Examples PCR primers are designed to:  Highly conserved DNA regions  Protein-coding regions with low degeneracy  More conserved regions that flank variable regions

6. Applications Primer design is used for:  Finding new genes  Developing new identification tools  Optimizing PCRs  Cloning  Sequencing,  DNA-based phylogeny,  functional analysis of genes etc….

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8. Primer Design Criteria

9. Primer Specificity Target: Conserved nucleotide or protein regions Primer length If the length is too short, it is difficult to design gene-specific primers and choose optimal annealing temperature. On the other hand, longer primers are more likely to form secondary structures that result in decreased PCR efficiency or promote primer dimer formation,, Usually 18 - 24 bases Base composition G+C content should be between 40% and 60%, with an even distribution of all four bases along the length of the primer. 9

10. End with 1-2 GC pairs, if possible,Primer 3’ end stability No inter- or intra-primer interactions Check with databases for specificity Cycling conditions and buffer concentrations should be adjusted for each primer pair (see PCR troubleshooting) Avoiding base run. 10

11. 11 Avoid sequence secondary structures Avoid complementary at 3` end of primers

12. 12 Primer melting temperature (Tm): Tm: is the temperature at which half the DNA strands are single stranded and half are double-stranded. The melting temperature (Tm) is the most important factor in determining the optimal PCR annealing temperature (Ta). Calculated Tm values of members of a primer pair should not differ by >5°C.

13. 13 Wallace rule: Tm = 4 * (G + C) + 2 * (A + T) Bolton and McCarthy: Tm = 81.5 + 16.6 * Log [I] + 0.41 * (%GC) – 600/L The nearest neighbor method (Santalucia et.al, 1998):

14.  Δ G (Gibbs free energy) is the most important factor that is to be taken into consideration in primer designing.  Δ G definition: The Gibbs Free Energy G is the measure of the amount of work that can be extracted from a process operating at a constant pressure. It is the measure of the spontaneity of the reaction.

15.  Primer secondary structures: Presence of the primer secondary structures adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.  Hairpins: A 3' end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3 kcal/mol is tolerated generally.  Larger negative value for ΔG indicates stable, undesirable hairpins. Presence of hairpins at the 3' end most adversely affects the reaction.  Self Dimer : A 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated generally. Rules for Primer designing…….

16.  Cross Dimer: Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.  3' End Stability : It is the maximum ΔG value (-8.5 kCal/mol is the default, however a value of -10 to -12kCal/mol is tolerated) of the five bases from the 3’.  An unstable 3' end (less negative ΔG) will result in less false priming. Primers with pentamer ΔG more stable than -12.0 kCal/mol (more negative) have a tendency to false prime and are more likely to form hairpins and self dimers. Rules for Primer designing…….

17. Primer Design Softwares 17

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20. 20 Gene of interest Mutation Detection Allelic discrimination Gene expression ( mRNA) Microbial agents detection Quantification … Disease

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