The researcher first PCR-amplifies the gene or gene segment of interest using a proofreading polymerase and primers that contain a specific 11-nucleotide sequence. The resultant product contains the gene of interest flanked on each end by a conserved, five-nucleotide topoisomerase-binding/cleavage site [(C/T)CCTT]. The six additional bases establish directionality for the subsequent topoisomerase-catalyzed joining step, in which sequence elements are specifically added to the 3' and/or 5' ends of the fragment.
Like a molecular crane, topoisomerase, covalently attached to the soon-to-be added sequence, conveys its cargo to the specified end of the PCR product. Topoisomerase first creates a single-strand nick in the PCR product, leaving an overhang that is compatible with its bound sequence element. The enzyme then uses its nucleic acid joining activity to fuse the element to the PCR product by joining the compatible ends. The resulting linear construct is then amplified via a secondary PCR reaction using element-specific ...
