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Background  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  Arabidopsis . The currently available methods for flanking sequence cloning, including the popular  TAIL-PCR  technique, are relatively laborious and slow.    Results  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. 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  FPNI-PCR  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  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 .    Conclusions  The successful amplification of target products through  FPNI-PCR  verified that this novel strategy is an effective, low cost and simple procedure. Furthermore,  FPNI-PCR  represents a more sensitive, rapid and accurate technique than the established  TAIL-PCR  and  hiTAIL-PCR  procedures.

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