A novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR.

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dc.contributor.authorAttatippaholkun, Watcharee Hen_US
dc.contributor.authorLek-ngam, Prapapanen_US
dc.contributor.authorBejrachandra, Sasitornen_US
dc.contributor.authorAttatippaholkun, Montree Ken_US
dc.date.accessioned2009-05-27T16:18:45Z
dc.date.available2009-05-27T16:18:45Z
dc.date.issued2002-03-18en_US
dc.descriptionThe Southeast Asian Journal of Tropical Medicine and Public Health.en_US
dc.description.abstractA novel molecular method for HIV-1 proviral DNA detection comprising two main techniques: nested PCR, amplifying a target sequence of the ENV-gene of HIV-1, and nonradioactively-reversed probe hybridization for the detection of the amplified target sequence. The dual amplification of inserted HIV-1 proviral DNA in each DNA sample to be tested was performed by nested PCR in two steps: firstly with two outer primers covering the target sequence of the ENV-gene of HIV-1; secondly with two 5'-biotinylated primers specific to the target sequence. The biotinylated PCR product could be visualized as a single band of 141bps in length on agarose gel stained with ethidium bromide. For the confirmation of the primary result, a method of reversed probe hybridization, using a nylon membrane immobilized with the oligonucleotide probe specific to the target sequence, was established. The oligonucleotide probe was given a homopolymer tail with terminal deoxyribonucleotidyl-transferase; the tail was spotted onto a nylon membrane and bound covalently by UV irradiation. Owing to its length, the tail bound to the nylon, leaving the oligonucleotide probe free to hybridize. Hybridization of the amplified target sequence to the immobilized probe was accomplished by a simple colorimetric reaction involving the enzymatic oxidation of a colorless chromogen that yielded a purple color wherever hybridization occurred.en_US
dc.description.affiliationDepartment of Clinical Chemistry, Faculty of Medical Technology, Siriraj Hospital Hospital, Mahidol University, Bangkok, Thailand.en_US
dc.identifier.citationAttatippaholkun WH, Lek-ngam P, Bejrachandra S, Attatippaholkun MK. A novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR. The Southeast Asian Journal of Tropical Medicine and Public Health. 2002 Mar; 33(1): 72-9en_US
dc.identifier.urihttps://imsear.searo.who.int/handle/123456789/34918
dc.language.isoengen_US
dc.source.urihttps://www.tm.mahidol.ac.th/seameo/2002_33_1/15-2856.pdfen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshDNA Probesen_US
dc.subject.meshDNA, Viral --analysisen_US
dc.subject.meshHIV-1 --geneticsen_US
dc.subject.meshHumansen_US
dc.subject.meshNucleic Acid Hybridizationen_US
dc.subject.meshPolymerase Chain Reaction --methodsen_US
dc.subject.meshProviruses --geneticsen_US
dc.subject.meshThailanden_US
dc.titleA novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR.en_US
dc.typeJournal Articleen_US
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