>>Published online before print August 8, 2002, 10.1073/pnas.162346699;
Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 17, 11073-11078, August 20, 2002
Biochemistry
A genotyping strategy based on incorporation and cleavage of chemically modified nucleotides
Jia Liu Wolfe,, Tomohiko Kawate, David A. Sarracino, Martin Zillmann, Jeffrey Olson, Vincent P. Stanton Jr., and Gregory L. Verdine§
Variagenics, Incorporated, 60 Hampshire Street, Cambridge, MA 02139; and § Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138
Communicated by David E. Housman, Massachusetts Institute of Technology, Cambridge, MA, June 10, 2002 (received for review November 9, 2001)
Aiming to facilitate the analysis of human genetic variations in the context of disease susceptibility and varied drug response, we have developed a genotyping method that entails incorporation of a chemically labile nucleotide by PCR followed by specific chemical cleavage of the resulting amplicon at the modified bases. The identity of the cleaved fragments determines the genotype of the DNA. This method, termed Incorporation and Complete Chemical Cleavage, utilizes modified nucleotides 7-deaza-7-nitro-dATP, 7-deaza-7-nitro-dGTP, 5-hydroxy-dCTP, and 5-hydroxy-dUTP, which have increased chemical reactivity but are able to form standard Watson-Crick base pairs. Thus one analog is substituted for the corresponding nucleotide during PCR, generating amplicons that contain nucleotide analogs at each occurrence of the selected base throughout the target DNA except for the primer sequences. Subsequent treatment with an oxidant followed by an organic base results in chemical cleavage at each site of modification, which produces fragments of different lengths and/or molecular weights that reflect the genotype of the original DNA sample at the site of interest. This incorporation and cleavage chemistry are widely applicable to many existing nucleic acid analysis platforms, including gel electrophoresis and mass spectrometry. By combining DNA amplification and analog incorporation into one step, this strategy eliminates preamplification, DNA-strand separation, primer extension, and purification procedures associated with traditional chain-termination chemistry and therefore presents significant advantages in terms of speed, cost, and simplicity of genotyping.
--------------------------------------------------------------------------------
To whom reprint requests should be addressed. E-mail: jwolfe@variagenics.com.<<
Cheers, Tuck |
