DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

11. pUC-SV: A New Double Adaptor Plasmid System for Sequencing Complex Genomes

Jonathan L. Longmire, Nancy C. Brown, Larry L. Deaven, and Norman A. Doggett

Bioscience Division and DOE Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87545

longmire@telomere.lanl.gov

The sequencing of complex genomes requires shotgun cloning (or subcloning) of genomic DNA (or BACs) into vectors that carry smaller inserts and that can serve as templates in sequencing reactions. Such subcloning vectors typically include plasmids or M13. For sequencing purposes at Los Alamos, we have previously used blunt end ligation of inserts into the HincII site of pUC-18. In addition, we have also used the double adaptor approach described by Andersson et al. ([1996] Analytical Biochemistry 236: 107-113) to subclone BAC fragments into pBluescript. Both of these approaches have distinct advantages and disadvantages. For example, blunt end subcloning is technically straight forward but can result in clones with multiple inserts and nonrecombinants even when vector ends are dephosphorylated. Double adaptor subcloning into pBluescript can reduce the frequency of nonrecombinants and clones with multiple inserts. However, the sequencing priming sites are located at a greater distance from the cloning site in Bluescript compared to pUC. Consequently, some of the sequence data that is generated in Bluescript clones is vector readthrough that has to be trimmed prior to assembly of the data.

In order to improve upon existing systems, we have developed a new cloning vector that allows double adaptor shotgun subcloning of large target molecules into pUC-18. The vector pUC-SV was constructed by cloning a 2 kb human DNA insert fragment into the XbaI and PstI sites of pUC-18. The insert serves as a "stuffer" and enables one to easily monitor for complete digestion when the plasmid is being processed to produce subcloning-ready vector. Vector adaptors are ligated to the SacI and SphI ends of the linearized vector. This produces 12 nt overhangs that are complimentary to adaptors that are ligated to the repaired ends of the fragmented target DNA. Nonrecombinants and clones with multiple inserts are eliminated because neither the vector adaptors nor the insert adaptors are self-complimentary. The pUC-SV vector yields cloning (subcloning) efficiencies greater than 105 colonies per microgram target DNA with zero nonrecombinant background. Highly representative subclone libraries can be made using as little as 10 ng of processed target DNA. In addition, the amount of DNA sequence data that is produced using pUC-SV is increased compared to Bluescript due to placement of the priming sites. In the adapted pUC-SV, the primer sites are located 26 nt and 28 nt away from the insert ends (compared to 53 and 64 nt in adapted Bluescript). Thus, 63 nt less data is lost to trimming for every subclone that is processed. This increased data yield becomes very significant when several thousands of subclones are processed.