BBF RFC 39 The USER cloning standard BBF RFC 39 : The USER cloning standard

0.4 Background and Motivation In living organism’s uracil DNA glycosylases (UDG) are used to prevent mutagenesis by eliminating uracil from DNA molecules by cleaving the N-glycosylic bond (Krokan et al., 2002). Uracil can be found in DNA due to misincorporation of dUMP residues or cytosine deamination (Lindahl, 1993). UDG is used in uracil-excision based cloning invented in the beginning of the 1990’s (Nisson et al., 1991). The general idea is to use a UDG and a DNA glycosylase-lyase Endo VIII to excise the uracil from the DNA to introduce single stranded overhangs on e.g. PCR products to allow cloning with long sticky ends. Both enzymes needed are included in the USER (Uracil-Specific Excision Reagent) enzyme mix from New England Biolabs.