One-Step PCR Sequencing. Final Technical Progress Report for February 15, 1997 - November 30, 2001

Abstract

We investigated new chemistries and alternate approaches for direct gene sequencing and detection based on the properties of boron-substituted nucleotides as chain delimiters in lieu of conventional chain terminators. Chain terminators, such as the widely used Sanger dideoxynucleotide truncators, stop DNA synthesis during replication and hence are incompatible with further PCR amplification. Chain delimiters, on the other hand, are chemically-modified, ''stealth'' nucleotides that act like normal nucleotides in DNA synthesis and PCR amplification, but can be unmasked following chain extension and exponential amplification. Specifically, chain delimiters give rise to an alternative sequencing strategy based on selective degradation of DNA chains generated by PCR amplification with modified nucleotides. The method as originally devised employed template-directed enzymatic, random incorporation of small amounts of boron-modified nucleotides (e.g., 2'-deoxynucleoside 5'-alpha-[P-borano]- triphosphates) during PCR amplification. Rather than incorporation of dideoxy chain terminators, which are less efficiently incorporated in PCR-based amplification than natural deoxynucleotides, our method is based on selective incorporation and exonuclease degradation of DNA chains generated by efficient PCR amplification of chemically-modified ''stealth'' nucleotides. The stealth nucleotides have a boranophosphate group instead of a normal phosphate, yet behave like normal nucleotides during PCR-amplification. The unique feature of our method is that the position of the stealth nucleotide, and hence DNA sequencing fragments, are revealed at the desired, appropriate moment following PCR amplification. During the current grant period, a variety of new boron-modified nucleotides were synthesized, and new chemistries and enzymatic methods and combinations thereof were explored to improve the method and study the effects of borane modified nucleotides on polymerase and unmasking mechanisms. This approach takes 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. In principle, the position of the modified nucleotide in each PCR product can be revealed in two ways, either by enzymatic unmasking (as previously described) or by chemical unmasking. We identified reagent sets for enzymatic or chemical conversion of boronated PCR products into mono- and bidirectional sequencing fragments. (a) We developed a new modified cytidine boranophosphate analogue that is (i) compatible with PCR, but more resistant to exonuclease III read-through than unmodified cytidine and (ii) permits better base calling; (b) We developed chemical methods for DNA and RNA cleavage at boronated nucleotide sites; and (c) We developed methods to quantify and detect stealth boranophosphate groups in DNA and RNA. Key advantages of boranophosphates as sequence delimiters in PCR are that they (1) delineate the DNA sequence yet (2) do not obstruct exponential amplification, and they (3) permit direct PCR sequencing, cycle sequencing, or RNA sequencing. They are also compatible with most sequencing platforms.