Five-color-based high-information-content fingerprinting of bacterial artificial chromosome clones using type IIS restriction endonucleases

We have developed a high-information-content fingerprinting (HICF) system f or bacterial artificial chromosome (BAC) clones using a Type IIS restrictio n endonuclease, HgaI, paired with a Type II restriction endonuclease, RsaI. In the method described, unknown five-base overhangs generated with HgaI a re partially or fully sequenced by modified fluorescent dideoxy terminators . Using an in-lane size standard labeled with a fifth dye, fragments are ch aracterized by both the size and the sequence of its terminal one to five b ases. The enhanced information content associated with this approach signif icantly increases the accuracy and efficiency of detecting shared fragments among BAC clones. We have compared data obtained from this method to predi cted HICF patterns of 10 fully sequenced BACs. We have further applied HICF to 555 BAC clones to assemble contigs spanning 16p11.2 to 16p13.1 of human chromosome 16. (C) 2001 Academic Press.