Direct PCR Sequencing with Boronated Nucleotides

A method is described to simultaneously amplify and sequence DNA using a new class of nucleotides containing boron. During the polymerase chain reaction, boron-modified nucleotides, i.e. 2′-deoxynucleoside 5′-α-[P-borano]-triphosphates, are incorporated into the product DNA. The boranophosphate linkages are resistant to nucleases and thus the positions of the boranophosphates can be revealed by exonuclease digestion, thereby generating a set of fragments that defines the DNA sequence. The boranophosphate method offers an alternative to current PCR sequencing methods. Single-sided primer extension with dideoxy-nucleotide chain terminators is avoided, with the consequence that the sequencing fragments are derived directly from the original PCR products. Boranophosphate sequencing is demonstrated with the Pharmacia and the Applied Biosystems 373A automatic sequencers, producing data that is comparable with cycle sequencing.