Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates.
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.
Nakamaye
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KL
Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, FRG.
Gish
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G
Eckstein
F
F
Vosberg
H P
HP
D001483:Base Sequence; D004270:DNA, Circular; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D005784:Gene Amplification; D013237:Stereoisomerism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D019914:Taq Polymerase
D004270:DNA, Circular; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D013438:Sulfhydryl Compounds; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase