Figure 2

A general (theoretical) scheme for FPNI-PCR (PCR based method for genomic walking or tagged flanking sequence cloning). In the first PCR step, single stranded copies of the target template are generated in the high stringency cycles, and double stranded products are produced in the low stringency cycle (in total, involving 3-5 repeated PCR cycles); in this primary step, amplification of the target products is likely to be accompanied with other, nonspecific, products. In the secondary and tertiary PCR steps (nested PCR), the target DNA is exponentially amplified by the gene specific and adaptor specific primers, while non-target genes are not amplified because there is no corresponding gene specific primer (and/or amplification was suppressed by the stem-loop structure of the DNA).