Method for purification and synthesis of RNA molecules and enzymatic RNA molecules in enzymatically active form.

Synthesis, deprotection, analysis and purification of RNA and ribozymes

The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.

Short cycle methods for sequencing polynucleotides

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5′-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo− mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F–A pair is associated with a brief pause, whereas that beyond A in the inverted A–F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A–F/F–A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

https://www.pnas.org/content/pnas/94/20/10506.full.pdf

A thymidine triphosphate shape analog lacking Watson–Crick pairing ability is replicated with high sequence selectivity

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

* To whom correspondence should be addressed. † Texas A&M University. Telephone: 979-845-4345. Fax: 979-8458839. E-mail: burgess@tamu.edu. ‡ ISIS Pharmaceuticals. Fax: 760-931-9200. E-mail: dcook@ isiph.com. Dan Cook, a keep-fit fanatic, was born in 1944 in the farm community of Clovis, NM. On his way to obtaining his doctorate in organic chemistry, under the direction of Raymond N. Castle, he attended the New Mexico Military Institute, Eastern New Mexico University, University of New Mexico, and Brigham Young University. He completed an industrial postdoctoral fellowship with Roland K. Robins at ICN Pharmaceutical in Costa Mesa, CA, in 1970−72, spent 11 years as a medicinal chemist at Warner-Lambert/ Parke-Davis Pharmaceutical Research Division in Ann Arbor, MI, and one year at Eastman Kodak Research Laboratories in Rochester, NY. After that he moved to Malvern, PA, with the Eastman Pharmaceutical Division/ Sterling Research Group. In 1989, he joined Isis Pharmaceuticals in Carlsbad, CA, where he is now Vice President, Research Chemistry. His research interests include heterocycles, nucleosides, nucleotides, and oligonucleotides as they relate to medicinal applications. Figure 1 Kevin Burgess is a professor at Texas A&M University, where he has been since September 1992. His research interest focuses on ways in which synthetic organic chemists can help to solve problems in medicinal chemistry and biotechnology. Specific research projects in The Burgess Group include peptidomimetics of protein−protein interactions, combinatorial chemistry, libraries of asymmetric catalysis, and methodology for high-throughput DNA sequencing. It is interest in the latter project that provided motivation to write this review. 2047 Chem. Rev. 2000, 100, 2047−2059

Lázló Ötvös

Papers

Simple synthesis of 5-vinyl- and5-ethynyl-2′-deoxyuridine-5′-triphosphates