Synthesis and Application of Charge-modified Dye-labeled Dideoxynucleoside-5'-triphosphates to 'direct-load' DNA Sequencing

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2002 Jun 28

PMID 12087172

Citations 3

Authors

Patrick J Finn

Lei Sun

Satyam Nampalli

Haiguang Xiao

John R Nelson

J Anthony Mamone

Greg Grossmann

Parke K Flick

Carl W Fuller

Shiv Kumar

Affiliations

Soon will be listed here.

Abstract

A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.

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