Figure 1. Schematic representation of LaNe RAGE. Dashed lines represent gDNA. Dotted lines represent DNA polymerization. Solid lines represent DNA formed during the previous step. 3' indicates the 3' end of DNA strands. The small box represents a known region of gDNA. The large box indicates intrinsic steps which take place during the first PCR of the LaNe RAGE procedure. Numbers 1 to 5 indicate gene-specific primer binding sites within a known region of gDNA. Underlined numbers indicate reverse complementary sequence relative to the initial gDNA strand. Number 6 indicates an optional gene-specific primer binding site for direct sequencing of product. Arrows indicate sites of primer binding. NNN3 labels the special hybrid primer used to prime initial DNA synthesis, consisting of a 5' gene-specific sequence (corresponding to position number 3) adjacent to 3' terminal degenerate, part-defined or defined sequence.

Initially, the NNN3 hybrid primer binds in a semi-random fashion at regular intervals along genomic DNA to prime DNA synthesis. During thermal cycling, gene-specific primer 1 binds at a specific site within the known region of the product of NNN3-primed extension to prime another extension reaction. This results in a product with gene-specific sequence at its 5' and 3' (corresponding to gene-specific sequence introduced by the hybrid primer) ends, with unknown sequence between. During thermal cycling, this is able to form a lariat structure, which primes intra-strand extension. The resultant product possesses 5' gene-specific sequence and extended 3' gene-specific sequence with unknown sequence between, which is amenable to two-sided gene-specific PCR series.