Kenneth W. Porter, J. David Briley, and Barbara Ramsay Shaw
Department of Chemistry, Duke University, Durham, NC 27708
A method to amplify and sequence DNA simultaneously by incorporating potential chain delimiters is described. During PCR amplification, a small percentage of boron modified nucleotides (e.g., 2'-deoxynucleoside 5'-[alpha]-P-borano-triphosphates1,2) are incorporated into the product DNA. The positions of the boranophosphates can be revealed by exonuclease digestion, thereby defining the sequence of the PCR product. The One-Step method improves current PCR sequencing methods by avoiding both DNA purification following amplification and single-sided primer extension with dideoxynucleotide chain terminators. As a consequence, One-Step sequencing is fast and amenable to automation. Data obtained by the One-Step method is comparable to that produced by cycle sequencing, yet requires much less DNA template.
A region of the p53 gene was sequenced by the One-Step boranophosphate method. The sequencing primer was modified with Cy5 and the resultant sequencing fragments were analyzed by an automatic fluorescent sequencer. As a comparison, the same region was sequenced by conventional cycle sequencing. For both the One-Step and cycle sequencing methods, the sequence could be read over approximately 450 bases. Sequence quality was similar for each method. In each case where a particular base could not be determined by one method, the base could often be called correctly by the other method. Therefore the One-Step method produces data that is comparable, as well as complementary, to dideoxy sequencing.
One-Step Sequencing should offer numerous advantages for DNA sequencing:
* Supported by NIH Grant HG00782 and DOE Grant DE-FG05-94ER61882.
[1] Sood, A., Shaw, B. R., and Spielvogel, B. F. (1990) J. Amer. Chem. Soc. 112, 90009001.
[2] Tomasz, J., Shaw, B. R., Porter, K., Spielvogel, B. F., and Sood. A. (1992) Angew. Chem. Int. Ed. Engl. 31. 1373-1375.