Producing DNA samples by shearing, nebulization, restriction enzyme digestion or PCR amplification frequently leaves DNA molecules with ends incompatible for downstream experiments. Selective enzymatic treatment is used to prepare DNA for ligation.
Ligation, the subsequent step to DNA end modification in the cloning process, is the formation of covalent phosphodiester bonds between the 3′ hydroxyl and 5′ phosphate ends of DNA and the vector. Preparation of DNA for vector ligation can include different types of end treatments, such as end-blunting, phosphorylation and dephosphorylation. Some enzymes couple the removal of the 3′ phosphate to expose hydroxyl groups, and the addition of a phosphate group to the 5′ end. Ligation of DNA with other molecules can either be through complementary sequence base pairing or blunt-ended ligation. The coupled action of enzymes can be used to remove any terminal single-stranded overhangs that are left after DNA digestion by restriction endonuclease or added to DNA during PCR amplification.
These end treatments can also be used to alter the phosphorylation state of the 5′ end of DNA for detection, isolation and sequencing applications. Many investigations use the addition of a phosphate radioisotope for labeling applications.
DNA End Modification includes these areas of focus:
Protocols for DNA End Modification
- Double Digest Protocol with Standard Restriction Enzymes
- End-labeling Protocol
- Loop-mediated Isothermal Amplification (LAMP)
- NEBNext End Repair Module Protocol (E6050)
- Non-radioactive Phosphorylation with T4 PNK or T4 PNK (3´ phosphatase minus)
- Optimizing Restriction Endonuclease Reactions
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Radioactive Labeling with T4 PNK or T4 PNK (3´ phosphatase minus)
- End-Modification Tips
Other Tools & Resources
Common Applications of Exonucleases and Endonucleases
Properties of Exonucleases and Endonucleases
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