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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
     
Restriction Enzyme Recognition Site
Acc65I G/GTACC
AclI AA/GCTT
AflII C/TTAAG
AluI* AG/CT
ApaI GGGCC/C
AvrII C/CTAGG
BamHI-HF G/GATCC
BglII A/GATCT
BsoBI C/YCGRG
BssSI C/ACGAG
CviAII C/ATG
CviQI* G/TAC
DpnII /GATC
EaeI Y/GGCCR
EcoRI HF G/AATTC
FatI /CATG
HaeIII* GG/CC
HindIII HF* A/AGCTT
HinfI G/ANTC
HpaI GTT/AAC
HphI GGTGA(N)8/
Hpy188I TCN/GA
Hpy188III TC/NNGA
HpyCH4V TG/CA
KpnI-HF GGTAC/C
MfeI HF C/AATTG
MscI TGG/CCA
MseI* T/TAA
NciI CC/SGG
NcoI HF C/CATGG
NlaIII CATG/
RsaI GT/AC
SacI HF GAGCT/C
SphI HF GCATG/C
TfiI G/AWTC
XhoI C/TCGAG
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