Richard A. Guilfoyle, Danhua Chen, Arthur Johnson, Todd Francisco, Tetsuyoshi Ono, David Rank and Lloyd M. Smith
University of Wisconsin, Dept. of Chemistry, Madison, WI 53706
We recently reported a direct selection strategy for shotgun cloning and sequencing in the bacteriophage M13 which utilized the vector called M13-100(1). Here, we describe the near-completed sequencing of a cosmid containing Drosophila genomic DNA using M13-102, a modified version of the vector. M13-102 contains two new additions: a homopurine- homopyrimidine tract for triplehelix-mediated affinity capture(2) (TAC), and the LacZ-derived universal primer sequences. TAC-purification of the linearized RF DNA is rapid - thus facilitating library construction, and specific - thus improving library quality by enabling efficient removal of non-vector DNA. Direct selection can (a) facilitate library production by eliminating the need for phosphatase treatment of the vector in lowering background, and (b) improve library quality by allowing phosphate treatment of the target DNA in order to reduce tandem insertion events. The cloning-efficiency analyses predicting these advantages are based on libraries constructed with a nebulizer-fragmented cosmid harboring the Drosophila GABA receptor gene. Preparation of the random single-stranded templates was performed using a streamlined solid-phase protocol developed in our laboratory and which is highly amenable to automation. Using the -21M13 dye-primers (Applied Biosystems, Inc.), fluorescent cycle-sequencing reactions were performed using Sequitherm[TM] (Epicentre Technologies, Madison, WI) and analyzed on ABI373A (non-stretch and stretch) machines. Based on the cosmid sequencing data, results will be presented describing the efficacy of the M13-102 strategy in terms (1) supporting the above predictions, and (2) analyses diagnostic for the production and sequencing of random clone libraries including contig assembly rates, redundancy, target coverage and gap-filling requirements.
1. Guilfoyle, R.A. and Smith, L.M. (1994) Nucleic Acids Research 22: 100-107.
2. Ji, H., Smith, L.M., and Guilfoyle, R.A. (1994) GATA 11: 43-47