Such as random priming or primer extension (using the Klenow fragment of Escherichia coli DNA polymerase I see Introduction: E. Radioactive nucleotides can be incorporated internally using one of several enzymatic methods, One 32P-dNTP and three unlabeled dNTPs, resulting in the incorporation of multiple 32P atoms per nucleic acid molecule. In internal labeling methods, radioactive derivatives of dNTP are substituted for their nonradioactive counterparts in reactions that are typically performed in the presence of This phosphorylation reaction results in the incorporation of one atom of 32P per nucleic acid molecule. Of DNAs and oligonucleotides, ATP is used to provide the 32P moiety, which is transferred to the 5′-most deoxyribonucleotide triphosphate (dNTP) using bacterial T4 polynucleotide kinase. In conventional in vitro radiolabeling, radioactive isotopes-usually 32P-are woven into the natural fabric of the probe in place of their nonradioactive homologs. Previous Section Next Section RADIOACTIVE VERSUS NONRADIOACTIVE LABELING OF NUCLEIC ACIDS Tables 1 and 2 summarize the options available for labeling nucleic acids and guide investigators to a method of labeling that suits the Success in all these applications depends on the facile introduction of label(s) into the nucleic acid or oligonucleotide. These techniques include Southern and northern analyses and in situ hybridization. Labeled DNA, RNA, LNA, and oligonucleotide probes are used in hybridization-based techniques to locate and bind DNAs and RNAs Labeled DNA and RNA can also be used as size markers in gel electrophoresis. In these cases, the labeled reagent isĬonverted to products of different sizes that can be detected by a variety of methods, the most common being gel electrophoresisįollowed by autoradiography or phosphorimaging. RNA, including digestion with S1 nuclease, RNase protection, and primer extension. Labeled fragments of cloned DNA and oligonucleotides of defined size are used as reagents in a variety of methods to analyze Labeled nucleic acids and oligonucleotides are used in molecular cloning either as reagents or as probes. Previous Section Next Section INTRODUCTION
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