Life Sciences
Primer-Dimer Formation: The Problem and the Solution Bassam El-Fahmawi, Ph.D. Axygen BioSciences
How Does Primer-Dimer Form? • Primer 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 Life Sciences
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Examples of Primer-Dimer Formation Intra
Inter
http://bioweb.uwlax.edu/genweb/molecular/seq_anal/primer_design/primer_design.htm Life Sciences
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Primer-Dimer Effect • Reduced amplification efficiency • Inaccurate quantification of PCR products and potentially misleading expression levels • Failure of amplification is dependent on downstream application sensitivity of primer dimer (e.g. NGS, Sanger Sequencing, and genotyping) • Inefficient use of amplification reagents, enzymes, labor, and time
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Chromatograms of Failed Sanger Sequencing Reactions
Lack of quality base calls
Overlying sequences
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How Can We Reduce the Potential for Primer-Dimer Occurrence? 1. Primer design 2. Optimization of amplification reaction recipe (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)
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Eight Tips for Primer Design 1. Primer Purity – 17-30 bases in length
2. Balance G/C and A/T-rich domains. – 20-70% G+C
3. Make 3’ end of primers a G, C, GC, or CG. – Or make GC content equal in both primers
4. Set melting temperature between 55-80°C 5. Avoid creating complimentary 3’-end base pairs 6. Avoid primer self-complementary 7. Avoid runs of three or more Cs or Gs at the 3’-end of primers 8. Use optimized concentrations of formamide, DMSO, Tween, or (NH4)2SO4 Life Sciences
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Primer Design: Software Approach There are several web-based, free primer-design software websites:
http://molbiol-tools.ca/PCR.htm Life Sciences
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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 Life Sciences
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I optimized every step in my PCR and I still get some primer-dimer artifacts in my reaction. Now what?
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Primer-Dimer Clean-up Strategies • Agarose gel requiring purification methods – Trough Method (polyethylene Glycol) – E-Gel® SizeSelect™ from Life Technologies™ – Band Excision from Gel
• Microfluids 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 • AxyPrep™ PCR Clean-up, AxyPrep™ DNA Gel Extraction Kit (Axygen) – Magnetic Beads-based Clean-up • AxyPrep™ Mag PCR Clean-up (Axygen)
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Agarose-based Gel Purification: Trough Method 1. Run your PCR product by electrophoresis (low-melting agarose). 2. Keep monitoring gel run until the desired separation pattern is 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 separation. Keep monitoring your band under UV until it flows through the trough. 5. Collect the PEG from the trough and continue using column-based clean-up glass milk or ethanol precipitation. Life Sciences
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Trough Method: Pros and Cons Pros -Efficient recover of primerdimer free PCR product
Cons -Multi-step procedure -Time consuming
-Inexpensive -Long UV exposure -Low throughput
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E-Gel® SizeSelect™ from Life Technologies™
No post clean-up required for the recovered PCR product
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-RNA-PurificationAnalysis/Nucleic-Acid-Gel-Electrophoresis/E-Gel-Electrophoresis-System/E-Gel-SizeSelect.html
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E-Gel® SizeSelect™: Pros and Cons Pros -Efficient recover of primerdimer free PCR product
Cons -Initial investment required -Closed system
-Convenient and simple -Low throughput -Limited yield
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Gel-based Purification: Two-step Process Step 1 Excise band from Gel with a razor
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Excise band from gel with Gel Cutting tips
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Gel-based Purification: Two-step Process Step 2: Extract DNA from gel slice with filtration column based kits
AxyPrep™ DNA Gel Extraction Kit (Axygen)
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Gel Excision Purification Method: Pros and Cons Pros -Efficient recover of primerdimer free PCR product
Cons -Multi-step procedure -Time consuming -Long UV exposure -Low recovery -Low throughput
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Microfluids Separation Systems: Pippen Prep™ from Sage Science
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Microfluids Separation Systems: LabChip® XT & XTe from Caliper/PE
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Microfluids Separation Systems: Pros and Cons Pros -Efficient recover of primerdimer free PCR product
Cons -Initial investment required -Limited yields
-Convenient and simple -Closed system -Low throughput
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Enzymatic-based Post PCR Clean-up Method: ExoSAP-IT® from Affymetrix®
Primer-Dimers
PCR products post ExoSAP-IT treatment
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ExoSAP-IT® Purification Method: Pros and Cons Pros -Fast post PCR clean-up method
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Cons -Inefficient primer-dimer removal method
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Filtration Column-based Clean-up Majority of the filtration column-based kits purify amplicons of 70 bp and higher
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Filtration Column-Based Purification Method: Pros and Cons Pros -Fast post PCR clean-up method
Cons -Efficiency of primer dimer removal depends on the PD size (>70 bp) -Not scalable for PD removal -Recovery efficiency depends on the size of the amplification product
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Magnetic Beads-based Clean-up Method
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Selection of the Proper Mag Beads Chemistry for Efficient Removal of Primer-Dimers or Adaptor Dimers
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No Visible Primer-Dimer Post Purification AxyPrep Mag PCR Clean-up
Agilent DNA 1000 Chip Electropherograms
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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
226
798
62,278
1.30%
• 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%)
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Mag Beads-Based Clean-up: Pros and Cons Pros
Cons
-Efficient and fast recovery of primer-dimer free PCR product
-Not manual-friendly
-Scalable
-Recovery efficiency depends on the size of the amplification product
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-Initial investment required
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Simplifying Mag Bead-based Clean-up IMAG: Magnetic-Beads Separation Devices Microplate
Tube 1. Load
3. Flip IMAG MSD to discard excess liquid
2. Separation 3060 Seconds
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Summary • Achieving amplification primer-dimer free depends on two key elements 1.Proper experimental design 2.Proper post-amplification clean-up strategy
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Thank You for Attending Today’s Seminar!
Any questions? My Contact information: Dr. Bassam El-Fahmawi
[email protected] www.axygen.com
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