Use of asymmetric PCR to generate long primers and single-stranded DNA for incorporating cross-linking analogs into specific sites in a DNA probe.

摘要: Photoactivatable DNA analogs have been incorporated enzymatically into and used to map the locations of polypeptides in protein complexes bound DNA. We developed a procedure for generating long primers from short oligodeoxyribonucleotides (oligos) incorporate cross-linkers at specific sites within either strand probes < or = 206 bp. Single-stranded molecules 52-206 nucleotides length were generated by asymmetric polymerase chain reactions (aPCR), using an excess one sense-strand primer be extended limiting amount each antisense that is complementary defines 3' end generated. The noncross-linking probe was also aPCR sequence interest. annealed full-length form partially double-stranded Cross-linking radioactive deoxyribonucleotides (dNTPs), followed normal dNTPs, onto cross-linking probes. This method reproducible avoids many difficulties encountered other published methods.