22. Direct Conversion of PCR Products into Bidirectional Sequencing Fragments 

Kenneth W. Porter, Ahmad Hasan, Kaizhang He, Jack Summers, and Barbara Ramsay Shaw 
Department of Chemistry, Duke University, Durham, NC 27708-0346 
brs@chem.duke.edu 

The search for more efficient and direct approaches to genomic sequencing continues to gain attention, particularly in gap-filling, finishing, and diagnostic applications. We have developed an alternate sequencing chemistry which avoids cycle sequencing, allows direct bidirectional genomic sequencing, and permits direct loading of PCR products onto the separating system. The method employs template-directed enzymatic, random incorporation of small amounts of boron-modified nucleotides (i.e. 2'-deoxynucleoside 5'-alpha-[P-borano]- triphosphates) during PCR amplification. The position of the modified nucleotide in each PCR product can be revealed in two ways, either enzymatically (as previously described¹) or chemically. Both approaches take advantage of differences in reactivity of the normal and modified nucleotidic linkages to generate PCR sequencing fragments that terminate at the site of incorporation of the modified nucleotide. By employing labeled PCR primers, the original PCR products are able to be converted directly into bidirectional sequencing fragments. 

In the enzymatic approach, the modification of a phosphate into a boranophosphate internucleotidic linkage prolongs its lifetime toward degradation by nucleases. The sequential hydrolysis by 3'-5' exonuclease III is thereby blocked by a boranophosphate, resulting in fragments that terminate in a boranophosphate nucleoside. However, normal and boranophosphate linkages with a 3'-cytosine are more susceptible to exonuclease degradation than other purines and pyrimidines, which reduces band uniformity. A series of base-modified cytosine derivatives were therefore synthesized and tested for nuclease resistance. The 5-ethyl-alpha-borano-dCTP analog was found to exhibit an increased resistance to exonuclease III compared to the alpha-borano-dCTP used previously in our method, without affecting incorporation, and resulted in more even banding patterns. Analysis with Basefinder software (M. Giddings) takes into account any mobility changes, permitting increased consistency and accuracy. The enzymatic approach may find use in applications where high resolution of longer fragments requires stronger signals at longer read lengths, because the distribution of fragments produced by nuclease digestion is skewed to long fragments. 

We are also developing a chemical method for generating sequencing fragments, as an alternative to exonuclease chew-back. In the chemical approach, we have identified reagents that selectively cleave the backbone of the PCR product at boranophosphate linkages, while leaving the normal phosphodiester linkages intact. We anticipate that chemical cleavage following incorporation of fluorescently labeled borano-dNTPs may result in a more efficient method of sequencing. Also under investigation are agents that can result in colorimetric detection of boranophosphate. 

Direct sequencing of PCR products simplifies mono- and bidirectional sequencing and provides a simple, direct, and complementary method to cycle sequencing. 

¹K.W. Porter, J. D. Briley, and B. R. Shaw, "One-Step PCR Sequencing with Boronated Nucleotides", Nucleic Acids Research 25, 1611-1617 (1997).