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 |
|
| 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
|
|
Strand targeted for removal requires one 5′ end with phosphate |
| 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 | |
| Progressive shortening of both ends of double-stranded DNA | Nuclease BAL-31 | |
| 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 |
