Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS.

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence.

DNAs with unusual properties revealed by field inversion gel electrophoresis of agarose-encapsulated DNA from mammalian cells.

Distinct DNA fractions (fr-DNAs), moving separately from bulk DNA, were revealed by field inversion gel electrophoresis of DNA from intact cells lysed and deproteinized in agarose plugs. These fr-DNAs (approximately 2% of the total DNA) were ubiquitously present in nuclei of all mammalian cells studied, including human normal and tumor tissues, and showed a typical electrophoretic pattern (three bands with constant mobilities termed a-, b-, and c-DNA). Characteristic mobility shifts induced by gamma irradiation of a- and b-DNAs suggest their non-linear conformation.