Restriction Enzymes for Droplet Digital PCR (ddPCR)

New England Biolabs (NEB) has evaluated a number of restriction enzymes for simple genomic DNA fragmentation in droplet digital PCR assays. This list is not meant to be exhaustive, but provides a starting point for validated enzymes with some simple usage guidelines. Each restriction enzyme listed below was successfully used for a ddPCR digest directly in the Bio-Rad® Droplet Digital SuperMix. The reaction was set up at room temperature with 200 ng human genomic DNA, and no additional incubation step or pre-emulsion inactivation.

Tips for Restriction Enzyme Selection

• Avoid enzymes blocked by CpG methylation
• Do not use an enzyme with a recognition site present in the target amplicon
• While fragment size does not impose strong limitations on enzyme choice, it is best to avoid targets containing fragments >50 kb
• If available, we recommend choosing the High Fidelity (HF®) version of an enzyme
• For digestion directly in the ddPCR reaction, we recommend choosing a Time-Saver™ Qualified enzyme

Restriction Enzymes in Droplet Digital PCR

Droplet digital PCR is a method for accurately quantitating copies of DNA or RNA in a sample. Each PCR reaction is separated into thousands or millions of droplets for analysis. 

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Protocol for Direct Digestion of gDNA for ddPCR

• Digestion is recommended whenever DNA input is greater than 75 ng
• Assemble ddPCR reactions at room temperature, this allows the restriction enzyme to digest the gDNA during the reaction set-up period
• Prepare reaction mixes as you would for a standard ddPCR reaction. Add 0.5–1 μL of each restriction enzyme (5–20 units, depending on enzyme concentration) to the reaction mixture
• After set-up, simply continue droplet generation as normal
• Restriction enzyme will be inactivated during first PCR denaturation step

Protocol for Digestion Prior to ddPCR

• Set-up restriction enzyme digests in the recommended NEBuffer
• Use 10 units of restriction enzyme per microgram of DNA sample
• Incubate for 5–60 minutes at the recommended reaction temperature for the respective enzyme
• Enzyme can be heat inactivated if desired, but this is not required
• No cleanup is necessary after digestion; sample can be directly added to ddPCR reactions
• Avoid carrying over more than a 1/10 amount of the restriction digest mixture to the ddPCR reaction