Fusion primer and nested integrated PCR (<it>FPNI-PCR</it>): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

<p>Abstract</p> <p>Background</p> <p>The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. 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 <it>Arabidopsis</it>. The currently available methods for flanking sequence cloning, including the popular <it>TAIL-PCR </it>technique, are relatively laborious and slow.</p> <p>Results</p> <p>Here, we report a simple and effective fusion primer and nested integrated PCR method (<it>FPNI-PCR</it>) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. 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). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall <it>FPNI-PCR </it>protocol. 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 <it>MYB </it>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 <it>Arabidopsis</it>.</p> <p>Conclusions</p> <p>The successful amplification of target products through <it>FPNI-PCR </it>verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, <it>FPNI-PCR </it>represents a more sensitive, rapid and accurate technique than the established <it>TAIL-PCR </it>and <it>hiTAIL-PCR </it>procedures.</p>