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Detection of Glucose 6-Phosphate Dehydrogenase by Bioluminescence-10

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Identifiers
DOI: 10.1016/b978-012426291-1/50012-1
Disciplines
  • Biology
  • Chemistry
  • Medicine

Abstract

Publisher Summary Nucleic acid hybridization has become a fundamental technique in biochemistry and molecular biology for detecting specific DNA sequences. Sensitive detection of the hybrid is obtained using probes labeled with radioisotopes, which unfortunately has a short half life and is not well suited to routine application. Other labels have been developed for this purpose, such as enzymatic incorporation of biotinylated or digoxigenin analogs, or chemical coupling of haptens, biotin, or enzymes. Some steps of this hybridization procedure can be eliminated by direct enzyme labeling of oligonucleotides; using luminescence as a detection system, several probes can be detected on the same filter. This chapter uses this strategy for the rapid identification of papillomaviruses after polymerase chain reaction (PCR). This technique can also be used for identification of different mutations or polymorphism in DNA sequences. However, if the number of variants is too high, as is the case for human leukocyte antigen (HLA) typing, identification can readily be accomplished by reverse hybridization between sequence-specific oligonucleotide probes immobilized on the filter and biotinylated amplified DNA samples. This process can be used to screen a sample for all known allelic variants at an amplified locus. The chapter develops a rapid bioluminescent assay that can be performed in tubes using an oligonucleotide immobilized on sepharose, and another labeled with glucose-6-phosphate dehydrogenase (G6PDH). This procedure is optimized to obtain high specificity, and it can be adapted for screening of numerous samples to identify pathogenic agents.

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