Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates.

Abstract:

:The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides and followed by treatment with an alkylating reagent that cleaves specifically at the phosphorothioate positions, is described. The Taq polymerase used in the amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-O-(1-thiotriphosphates) as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed with either 2-iodoethanol or 2,3-epoxy-1-propanol. The higher reactivity of 2,3-epoxy-1-propanol allows less reagent to be used to obtain the same amount of degradation as with 2-iodoethanol.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Nakamaye KL,Gish G,Eckstein F,Vosberg HP

doi

10.1093/nar/16.21.9947

subject

Has Abstract

pub_date

1988-11-11 00:00:00

pages

9947-59

issue

21

eissn

0305-1048

issn

1362-4962

journal_volume

16

pub_type

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