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

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 http://bioweb.uwlax.edu/genweb/molecular/seq_anal/primer_design/primer_design.htm

Examples of Primer-Dimer Formation Intra Inter http://bioweb.uwlax.edu/genweb/molecular/seq_anal/primer_design/primer_design.htm

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.

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

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).

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) 

Primer Design: Software Approach Several web-based free access software sites http://molbiol-tools.ca/PCR.html

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

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

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

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

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

E-Gel SizeSelect from Life Technologies http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-RNA-Purification-Analysis/Nucleic-Acid-Gel-Electrophoresis/E-Gel-Electrophoresis-System/E-Gel-SizeSelect.html No post clean-up required for the recovered PCR product

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

Microfluidics Separation Systems: Pippin from Sage Science Size Selection Range Agarose %     For Ranges Between 2 0.1 - 0.6 kb 1.5 0.3 - 1.5 kb 50-100 Minutes Run

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

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

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

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

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

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

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

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

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

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

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

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

Read Length Distribution after GS FLX Sequencing Run Post AxyPrep Mag PCR Clean-up Sample 66 bp fragment 98 - 104 bp fragments 50 - 150bp 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%)

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

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 30-60 Seconds

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

Questions? Thank you