March 18, 2021

PCR reagent selection: what, why and which?

By Cytiva

The success of PCR depends on making suitable choices for the approach, design, reagents, and instruments you use. Here we provide a quick guide to reagent options, and the challenges they address.

Despite many advances in nucleic acid amplification tests (NAATs), polymerase chain reaction (PCR) still lies at the heart of every laboratory that works with nucleic acids. From basic to applied research, PCR helps identify, sequence, or modify RNA and DNA.

But even this well-established method can go wrong. PCR can create non-specific, false positives, heterogeneous products with different genetic mutations, and sometimes no product at all. How can you minimize these challenges in your work?

Use this handy chart to understand the roles that different reagents play, the challenges they can address, and options to help you achieve PCR success.

Reagents for PCR

Reagent

Function

Challenges

Solutions

Primers

 Bind to specific sequences of denatured DNA flanking target DNA.
Bind in a 5’ to 3’ and 3’ to 5’ direction.		 • Self-anneal
• Primer-dimer formation
• Different Tm values
• Non-specific DNA amplification in RNA analysis 		• Use software or online tools to aid design
• Re-design primers with:
• low GC content
• fewer nucleotide repeats
• similar Tm values
• Design primers across intron-exon boundaries or exon-exon junctions

Probes

 Bind to denatured DNA in a target-specific manner and are fluorescently labelled (used in qPCR). • Non-specific binding
• No binding
• No fluorescence
• Bleeding of dyes in multiplex reaction		 • Re-design probes with:
• low GC content
• higher Tm (6-8oC) to primers
• Choose different type of probe
• Chose fluorescent detection markers that have separate emission properties

DNA polymerase

 Catalyzes the synthesis of DNA from dNTPs. • Low fidelity-lacks 3’ to 5’ exonuclease or ‘proofreading’ activity
• Generation of partial products		 • Choose high-fidelity enzyme
• Use ‘hot start’ enzyme to avoid activation at sub-optimal temperatures
• Optimize elongation temperatures

Buffer

 Provide a stable chemical environment for DNA polymerase activity. • Reduced DNA polymerase activity • Optimize the pH for specific DNA polymerase
• Add individual ions to alter pH, e.g., Mg2+

dNTPs

 Provide the building blocks for DNA synthesis. • PCR inhibition • Optimize concentration of dNTPs – excessive dNTPs can lead to inhibition

Additives

Increase yield, specificity, and consistency of PCR. • Secondary structures created by high GC content
• PCR inhibition		 • Addition of enhancers can help with challenging PCR conditions
• Replacing dGTP with 7-deaza dGTP, adding DMSO or formamide will reduce secondary structures
• Addition of BSA can overcome PCR inhibition

At Cytiva, we provide a broad range of tools and solutions for DNA sample preparation and PCR. Visit our genomics blogs for news, tips, and insights. For support in any aspect of your workflow, contact our Scientific Support team.

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