Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside α-thiotriphosphates
- 1 January 1988
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 16 (21) , 9947-9959
- https://doi.org/10.1093/nar/16.21.9947
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 degredation 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.This publication has 15 references indexed in Scilit:
- Direct Detection of HIV-1 RNA from AIDS and ARC Patient SamplesDNA, 1988
- Genomic Amplification with Transcript SequencingScience, 1988
- Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA PolymeraseScience, 1988
- Protection of oligonucleotide primers against degradation by DNA polymerase IBiochemistry, 1987
- Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic SequencesScience, 1986
- Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell AnemiaScience, 1985
- NUCLEOSIDE PHOSPHOROTHIOATESAnnual Review of Biochemistry, 1985
- The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNANucleic Acids Research, 1985
- The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNANucleic Acids Research, 1985
- DNA sequencing with chain-terminating inhibitorsProceedings of the National Academy of Sciences, 1977