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DNA and RNA sequencing utilizing phosphorothioate chemistry.

G Gish¹, F Eckstein

¹Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, FRG.

Nucleic Acids Symposium Series

|January 1, 1987

Summary

A novel nucleic acid sequencing method utilizes phosphorothioate diesters and 2-iodoethanol for DNA and RNA analysis. This approach enables efficient sequencing through specific chemical cleavage and subsequent electrophoresis.

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Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Nucleic acid sequencing is fundamental to molecular biology.
Existing sequencing methods have limitations in efficiency and chemical treatment.
Phosphorothioate analogs offer unique chemical properties for modification.

Purpose of the Study:

To develop a novel method for nucleic acid sequencing.
To utilize the specific hydrolysis of phosphorothioate diesters for sequencing applications.
To adapt the method for both DNA and RNA sequencing.

Main Methods:

DNA sequencing involves polymerizing primed single-stranded M13 DNA with Klenow fragment and alpha-phosphorothioate deoxyribonucleotide triphosphate.
RNA sequencing uses alpha-phosphorothioate ribonucleotide triphosphates during in vitro transcription.

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Both methods are followed by chemical treatment with 2-iodoethanol or iodoethane, precipitation, and polyacrylamide gel electrophoresis.

Main Results:

The method demonstrates successful sequencing of both DNA and RNA molecules.
Cleavage occurs via alkylation of the sulfur atom in the phosphorothioate linkage, followed by hydrolysis.
The process allows for the separation and analysis of resulting DNA fragments.

Conclusions:

A new chemical method for nucleic acid sequencing has been established.
The technique offers an alternative approach for DNA and RNA sequence determination.
This method leverages the controlled chemical cleavage of phosphorothioate linkages.