A 3´→ 5´ proofreading exonuclease domain is intrinsic to most DNA polymerases. It allows the enzyme to check each nucleotide during DNA synthesis and excise mismatched nucleotides in the 3´ to 5´ direction. The proofreading domain also enables a polymerase to remove unpaired 3´ overhanging nucleotides to create blunt ends. Protocols such as high-fidelity PCR, 3´ overhang polishing and high-fidelity second strand synthesis all require the presence of a 3´→ 5´ exonuclease.
In contrast, some applications are enhanced by the use of polymerases without proofreading activity. For example, the efficiency of DNA labeling is enhanced by the absence of proofreading, because it prevents the excision of incorporated bases, allowing for the use of less of the modified base.
Modified base incorporation assays, such as multicolor analysis of gene expression, gene mapping, and in situ hybridization, which utilize DNA that has been labeled with a fluorescent nucleotide to facilitate detection, are well matched to NEB's exonuclease-deficient DNA polymerases. Non-proofreading polymerases are also indispensable when partially filling in 5´ overhangs with only selected dNTPs. Addition of an untemplated dNTP at the 3´ terminus of blunt ends, a requirement for TA cloning, is also promoted by non-proofreading enzymes. In addition to several wild type polymerases in each of these categories, NEB offers genetically altered versions of several proofreading polymerases, where the proofreading exonuclease activity has been attenuated or abolished.