Common Applications for Exonucleases and Endonucleases

Application Recommended Enzyme(s) Notes
Removal of 3′ overhangs Quick Blunting Kit  
5′ overhang Fill in Treatment Quick Blunting Kit  
Removal of single-stranded primers for nested PCR reactions Thermolabile Exonuclease I   
Removal of primers post PCR prior to DNA sequencing or SNP detection Exonuclease I
Thermolabile Exonuclease I 
Exonuclease VII 

Quick Heat inactivation versus Exonuclease I
For 3′ chemically modified primers

Mapping positions of introns in genomic DNA Exonuclease VII    
Removal of primers with or without 3′ or 5′ terminal phosphorothioate bonds Exonuclease VII    

Generating ssDNA from linear dsDNA:

If 5′ → 3′ polarity required

If 3′ → 5′ polarity required


Lambda Exonuclease 

Exonuclease III 


Strand targeted for removal requires one 5′ end with phosphate

Strand targeted for removal requires a 5′ overhang, a blunt end, or a 3′ overhang (with less than 4 bases)

Preparation of nested deletions in double-stranded DNA Exonuclease III (E. coli) plus Exonuclease VII    
Site-directed mutagenesis Exonuclease III (E. coli) 

T7 Exonuclease 
Removes nicked-strand DNA from 3′ to 5′

Removes nicked-strand DNA from 5′ to 3′
Nick-site extension T7 Exonuclease    
Degradation of denatured DNA from alkaline-based plasmid purification methods for improving DNA cloning T5 Exonuclease   
Removal of chromosomal/linear DNA in plasmid preparations T5 Exonuclease

Exonuclease V (RecBCD)
Degrades linear ss + dsDNA, nicked DNA

Degrades linear ss + dsDNA:  PREFERRED as Exo V will save nicked plasmids resulting in higher yields especially for low-copy number plasmid prep
Removal of unligated products (linear dsDNA) from ligated circular double-stranded DNA T5 Exonuclease

Exonuclease V (RecBCD) 
Only the un-nicked form of ligated circular double-stranded remains

Both nicked and unnicked-form of ligated circular double-stranded DNA remains
Removal of residual gDNA after purification of low copy plasmid Exonuclease V (RecBCD)   
Removal of contaminated  DNA from RNA samples DNase I  
Conversion of single-stranded DNA  or RNA to 5′-mononucleotides Nuclease P1   
Analysis of base composition, potential damage and modification of nucleic acids Nuclease P1    
Preparation of double-stranded DNA fragments with 5′-OH and 3′-phosphate Micrococcal Nuclease   
Degradation of nucleic acids (both DNA and RNA) in crude cell-free extracts Micrococcal Nuclease   
Preparation of rabbit reticulocyte Micrococcal Nuclease   
Chromatin Immunoprecipitation (ChIP) analysis Micrococcal Nuclease