Methods for rapid cloning and detection for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known sequences in bacterial chromosomes

Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. Howeve r, the protocols for inverse PCR introduced so far consist of some time-con suming procedures, and with them, we cannot "walk" chromosomes too far sinc e the number of suitable restriction enzymes is limited. Our experiments le d to confirming simpler technical approaches applicable to the case of bact erial chromosomes, that is, designing two end-specific "contextual" sequenc es with which we can quickly detect the desired clones of targeted DNA frag ments by simply analyzing PCR products, employing "the minimum value of the desired fragments" as a "discriminating minimum" value to decrease contami nant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence ("reordering") for PCR amplification in combination wit h cloning of the inverse PCR-generated DNA, With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR i n "walking" chromosomes.