END SEQUENCE DETERMINATION FROM LARGE INSERT CLONES USING ENERGY-TRANSFER FLUORESCENT PRIMERS

Genome mapping strategies depend heavily on confirmatory data of sever al types to establish overlaps between contiguous stretches of cloned DNA derived from genomic regions. One type of ancillary data that can contribute to establishing these overlaps is DNA sequence data derived from the ends of large (>30 kb) inserts in genomic clones. This type of data can be difficult to obtain routinely, because large clones are often unstable and microgram quantities of highly purified DNA are re quired in each sequencing reaction to obtain sufficient signal for acc urate base calling and maximum read length. Recently, we have been exp erimenting with methods to consistently obtain up to 800 bases of high -quality sequence data from the ends of large insert clones using Ther moSequenase DNA polymerase and Energy Transfer fluorescent primers. Ou r experimental approach and results, described in this paper, indicate that routinely obtaining high-quality sequence data from the ends of large insert genomic clones is feasible. Such data can contribute to t he assessment of common regions between large insert clones, to the es tablishment of conservation of synteny between closely related species , and to the detection of additional contiguous clones.