Identification and characterization of LA08NC01 cosmids containing rare cutter AscI sites

LA08NC01 is a flow-sorted human chromosome 8 cosmid library that was constructed and arrayed at Los Alamos. We have used this library to produce a sub-library of those cosmids containing AscI (GGCGCGCC) sites, which are therefore AscI-linking clones. Two protocols have been employed to identify AscI sites. The first protocol relies upon restriction digestion for cloning into doubly digesting plasmids and thereby recovering an end clone. The second protocol relies upon sequence directly, by using a 12-mer NNNNGGCGCGCC as a DNA hybridization probe. Using these protocols we have identified and confirmed 44 cosmids that contain AscI sites. Our goal is to develop markers that are rich in information. Consequently, these cosmids have been screened for CA repeats, which provide a polymorphic STS. The region surrounding the AscI site has been sequenced to provide an identifier and developed as an STS site for those cosmids lacking a CA repeat. The sequence identifier has been used for sequence database library searches. We have identified 3 genes from 8p after screening the identifiers for 10 cosmids. In addition, we have found 2 additional AscI sites from the known genes SFTP2 and POLB. Identification of the AscI site adjacent to POLB required a chromosomemore » walk of 2 steps. Many of these cosmids are rich in information since they are frequently polymorphic, contain STS sites, provide linking clones for PFGE mapping, and often encode genes that may be placed on expression maps. In conclusion, while the total number of identified cosmids is small, the majority of them are extremely rich in information.« less