1. Primer-Dimer Formation: The Problem and the Solution
Bassam El-Fahmawi, PhD

2. How Does Primer-Dimer Form?
Primer inter and intra interactions that give rise to non-specific hybridized by-products
Less primer available for the amplification reaction

3. Examples of Primer-Dimer Formation
Intra
Inter

4. Primer-Dimer Effect Reduced Amplification efficiency.
Inaccurate quantification of PCR products and potentially misleading expression levels.
Failure of amplification dependent downstream application (e.g. NGS, Sanger Sequencing, genotyping).
In efficient use of amplification reagents, enzymes, labor and time.

5. Lack of quality base calls
Chromatograms of Failed Sequencing Reactions
Lack of quality base calls
Overlying sequences

6. How Can we Reduce the Potential for Primer-Dimer Occurrence?
1- Primer design.
2- Optimization of amplification reaction reciepie (Taq, dNTPs, primer, Mg++).
3- Amplification enzyme selection (e.g. Hot start or Taq designed for GC rich regions).
4- Amplification cycling conditions optimization (e.g. melting and annealing temperatures).

7. Tips for Primer Design
1.  Primers should be as pure as possible17-30 bases in length.  2.  base composition should be 20-70% (G+C);  Whenever possible, avoid an unbalanced distribution of G/C- and A/T-rich domains. 3.  primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;   or try to make the GC content equal in both primers. 4.  Melting temperature between 55-80◦C reduces the occurrence of hairpins; 
5.  3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesized preferentially to any other product;   6.  primer self-complementarity (ability to form secondary structures such as hairpins) should be avoided;   7.  Runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.  
8. Use optimized concentration of formamide or DMSO or Tween or (NH4)2SO4.
  (adapted from Innis and Gelfand,1991) 

8. Primer Design: Software Approach
Several web-based free access software sites

9. Primer-dimer range can vary from 50 bp up to 150 bps
Primer-Dimer Detection
Primer-dimer range can vary from 50 bp up to 150 bps
Primer-dimer
Real-time cycler (melting curve)
Gel Electrophoresis
Agilent DNA 1000 Chip electropherograms

10. Optimized Every Step in my PCR and I Still Get Some Primer-Dimers Artifacts in my Reaction, Now What?

11. Primer-Dimer Clean up Strategies
Agarose gel requiring purification methods
Trough Method (polyethylene Glycol)
E-gel SizeSelect from Life Technologies
Band Excision from Gel
Microfluidics Separation Systems
LabChip XT (Caliper/PE) and Pippin Prep (Sage Science)
Non Agarose gel requiring purification methods
Enzymatic based Post PCR Clean up
Exonuclease I and Shrimp Alkaline phosphate cocktail or ExoSAP-IT (Affymetrix)
Filtration Column-based Clean up
Magnetic beads-based cleanup

12. Agarose based Gel Purification: Trough Method
1- Run your PCR product by Electrophoresis (low melting agrose)
2- Keep monitoring your gel run until the desired separation pattern obtained
3- Take the gel out of the running tank. Make an excision in the gel to make the trough, then fill it with 20% of PEG 8000.
4- Return the gel to the running tank and continue your separation. Keep monitoring your band under UV until it flows in the trough.
5- Collect the PEG from the trough and continue using column based clean up glass milk or ethanol precipitation

13. Trough Method: Pros and Cons
Multi-step procedure
Time consuming
Long UV exposure
Low throughput
Efficient recovery of primer-dimer free PCR product
Inexpensive

14. E-Gel SizeSelect from Life Technologies
No post clean-up required for the recovered PCR product

15. E-Gel SizeSelect: Pros and Cons
Efficient recovery of primer-dimer free PCR product
Convenient and simple
Initial investment required
Closed system
Low throughput

16. Microfluidics Separation Systems: Pippin from Sage Science
Size Selection Range
Agarose %
    For Ranges Between 2 kb
1.5 kb
Minutes Run

17. Microfluidics Separation Systems:
LabChip XT & XTe from Cliper/PE

18. Microfluidics Separation Systems : Pros and Cons
Initial investment required
Low yields
Closed system
Low throughput
Efficient removal of a primer or adapter-dimer
Convenient and simple

19. Gel Based Purification: Two Step Process
Step I
Excise band from Gel with a razor
Excise band from gel with Gel Cutting tips or

20. Extract DNA from gel slice with filtration column based kits
Step II
Extract DNA from gel slice with filtration column based kits

21. Gel Excision Purification Method: Pros and Cons
Multi-step procedure
Time consuming
Long UV exposure
Low Recovery
Low throughput
Efficient recovery of a primer-dimer free PCR product

22. PCR products post ExoSAP-IT treatment
Enzymatic based Post PCR Clean up Method: ExoSAP-IT * * *
Primer -dimer
PCR products post ExoSAP-IT treatment

23. ExoSAP-IT Purification method: Pros and Cons
Fast post PCR clean up method
In efficient primer-dimer removal method

24. Primer-dimer range can vary from 50 bp up to 150 bps
Filtration Column-based Clean up
Majority of the filtration column based kits purify amplicons of 70 bp and higher
Primer-dimer range can vary from 50 bp up to 150 bps
PCR pre clean up
PCR post clean up PD
Primer-dimer around 85 bp

25. Filtration Column-Based Purification Method: Pros and Cons
Efficiency of primer dimer removal depends on the PD size (>70 bp)
Not scalable for PD removal
Fast post PCR clean up method

26. Magnetic Beads-Based Clean up Method
Three Basic Steps
Wash
Bind
Elute

27. Selection of the proper Mag beads Chemistry for Efficient Removal of Primer-Dimers or Adaptor-Dimers
Mag bead based
Column Based
AxyPrep
Ampure XP
MinElute
Illumina Library Clean-up Post Ligation

28. No Visible Primer-Dimer Post Purification
AxyPrep Mag PCR Clean-up
Pre
Sample 1
Sample 2
Post
Agilent DNA 1000 Chip electropherograms

29. Read Length Distribution after GS FLX Sequencing Run
Post AxyPrep Mag PCR Clean-up
Sample
66 bp fragment
bp fragments
bp fragments
Total # of Reads
% short reads
Mag_Pro81_B4
226 37
686
55,598
1.20%
Mag_Pro81_C7
293 29
831
46,670
1.70%
Individual_Pro81 20
187
660
64,240
1.00%
Individual_H+B 6
143
545
59,694
0.90%
Individual_Pro518 69
798
62,278
1.30%
The data indicates that the percent of total number of reads attributed to reads less than 150bp in length
Sequencing data indicate that removal of short fragments is not 100% effective; however, the number of short fragments present in the total read population is extremely low (no more than 1.7%)

30. Mag Beads-Based Clean up Method: Pros and Cons
Not manual friendly
Initial investment required
Recovery efficiency depends on the size of product
Efficient and fast recovery of primer-dimer free PCR product
Scalable

31. Simplifying Mag Bead-Based Clean up
IMAG: Magnetic-Beads Separation Devices
Microplate
Tube
3- Flip IMAG MSD to discard excess liquid
1- Load
2- Separation Seconds

32. Summary
Achieving amplification primer-dimer free depends on two key elements:
1- Proper experimental design
2- Proper post amplification clean-up strategy

33. Questions?
Thank you