Figure 1

Schematic overview of the amplification of an oligonucleotide contained within a suicide cassette. (A) A DNA sequence comprising an oligonucleotide to be amplified (red) and a suicide cassette (blue) is circularized by self-templated hybridization in the suicide cassette region. (B) The DNA circle is closed by self-templated ligation. (C) By the addition of a primer, complementary to part of the DNA sequence, dNTPs and a polymerase a rolling circle DNA synthesis can be initiated resulting in tandem repeats complementary to the templating circle. (D) The suicide cassette can be removed from the oligonucleotide by cleavage with the Mly I restriction enzyme. (E) The tandem repeats can be linearized without the loss of nucleotides using the Nt. Alw I nicking enzyme, resulting in monomers complementary to the initial DNA sequence. (F) Self-templated hybridization (see step A). (G) Self-templated ligation (see step B). (H) Rolling circle DNA synthesis (see step C). (I) Cleavage with Mly I (see step D). (J) Nicking, using Nt. Alw I, resulting in monomers of the same polarity as the initial DNA sequence. (+) and (-) indicate the polarity of the oligonucleotide.