The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. Here, we report a simple and effective fusion primer and nested integrated PCR method ( FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. The advent of genomics-based technologies has revolutionized many fields of biological enquiry.

Create primers in serial cloner