Schematic of Nested Patch PCR. (A) A PCR reaction containing primers pairs for all targets is performed on genomic DNA. The primers contain uracil substituted for thymine. The primers are then cleaved from the amplicons by the addition of heat-labile uracil DNA glycosylase, endonuclease VIII, and single-strand-specific exonuclease I. (B) The ends of the target regions are now internal to the PCR primers (nested). (C) Nested Patch oligonucleotides are annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers. The universal primers are then ligated to the amplicons. The universal primer on the 3′ end of the amplicon is modified with a three carbon spacer that protects the selected amplicon from the final exonuclease reaction that degrades nonspecific products. (D) The selected amplicons are then amplified together simultaneously by PCR with universal primers.
