DOE Human Genome Program Contractor-Grantee
16. Chemical Conversion of Boronated PCR Products into Bidirectional Sequencing Fragments
Barbara Ramsay Shaw, Kenneth W. Porter, Ahmad Hasan, Kaizhang He, and Jack Summers
Department of Chemistry, Duke University, Durham, NC 27708-0346
We 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 boronated 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 nucleoside boranophosphate. However, normal and borano-phosphate 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. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhances the cytidine boranophosphate resistance towards exonuclease III (i.e., 5-ethyl-dC > 5-methyl-dC > dC 5-bromo-dC > 5-iodo-dC). The best analog, 5-ethyl-a-borano-dCTP, not only showed an increased resistance to exonuclease III compared to the a-borano-dCTP used previously in our method, but did so without affecting incorporation and resulted in more even banding patterns2. 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.
In the chemical approach, we have examined several methods for generating sequencing fragments, as an alternative to exonuclease chew-back. First, we identified reagents that selectively cleave the backbone of the PCR product at deoxy boranophosphate linkages, while leaving the normal phosphodiester linkages intact. Second, we synthesized a new boranophosphate RNA dimer analogue3 and found conditions under which the ribo boranophosphate linkage is considerably more susceptible to cleavage than a deoxy or normal phosphodiester linkage. We then synthesized diastereomers of ribonucleoside 5'-(a-P-borano)triphosphates4 and showed that one isomer can be incorporated readily into RNA with T7 RNA polymerases, yielding boronated transcripts that are thousands of nucleotides long. We are now examining DNA polymerases that can incorporate the boronated RNA triphosphates into DNA. Also under investigation are agents that can result in colorimetric detection of boranophosphate. Direct sequencing of PCR products by cleavage of boranophosphates should simplify mono- and bidirectional sequencing and provide a simple, direct, and complementary method to cycle sequencing.
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