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

13. An Isothermal Amplification System for the Production of DNA Templates for DNA Sequencing

Stanley Tabor and Charles Richardson

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

stabor@hms.harvard.edu

We are developing DNA polymerases for use in DNA sequencing and amplification applications. We will describe a very efficient isothermal amplification system which provides an attractive alternative to conventional methods of generating plasmid and BAC templates for DNA sequencing. Amplification of from 1 pg to 1 µg of template DNA results in the synthesis of DNA to a final concentration of 0.5 µg/µl in 15 min at 37 °C (corresponding to an amplification of up to several million-fold). Amplification is nonspecific; all sequences present are amplified equally. The reaction requires no exogenous primers. This system is based on the replication apparatus of bacteriophage T7; the principle enzymes required are two forms of T7 DNA polymerase, the T7 helicase/primase, and single-stranded DNA binding protein. The products are linear double-stranded DNA fragments several thousand base pairs in length. When the products are used as templates for capillary-based fluorescent sequencing, the fluorescent signal produced is several fold higher than a comparable amount of supercoiled plasmid DNA, and results in 20% more base calls that have a quality score greater than Phred 20. The attractive features of this system for large sequencing projects is its simplicity and the constant, reproducibly high yield of DNA that can be used directly in DNA sequencing reactions without further purification. This nonspecific amplification reaction could also be of use in immortalizing small, precious samples of genomic DNA required for genotype analysis. We are also using this technology to amplify single DNA molecules embedded in agarose. This enables one to construction and amplification DNA libraries in vitro without the need to transform bacterial cells. Finally, we will present an update of our work modifying DNA polymerases to increase their processivity and their use of nucleotide analogs for use in DNA sequencing.