Practice question

Inverse PCR enables amplification of sequences flanking a known region without prior knowledge of adjacent DNA. Genomic DNA is digested with restriction enzymes that cut outside the known segment, then circularized via dilute ligation favoring intramolecular joining. Primers designed in outward orientation relative to the conventional arrangement, facing away from the known core, now point toward each other on the circular template and amplify the unknown surrounding regions. Nested, touchdown or colony PCR serve different purposes like increasing specificity, optimizing annealing or screening bacterial colonies and cannot recover flanking unknowns. Inverse PCR is thus vital for mapping insertions and promoters.