Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.
INTRACELLULAR AMPLIFICATION OF THE PROVIRAL DNA OF MOUSE MAMMARY TUMOR VIRUS USING THE POLYMERASE CHAIN REACTION
Diss Abstr Int [B]; 52(3):1232 1991. Unique Identifier : AIDSLINE ICDB/92678997 Chiu K; Univ. of California, Davis
Abstract:
The purpose of this research is to develop a technique for amplifying cellular DNA in situ using the polymerase chain reaction (IS-PCR), which has the potential to be a powerful method for pathogenesis studies. We adopted mouse mammary tumor virus (MMTV) as the model system. MMTV is a retrovirus that induces tumors in the mouse mammary gland. Retroviruses have been intensively studied since the discovery in 1971 of their unique enzyme reverse transcriptase which reverses the flow of genetic information from RNA to DNA. Recent studies on the human immunodeficiency virus revealed another remarkable property of retroviruses: A highly complicated regulatory mechanism functions between the virus and its host resulting in a complicated pathogenesis pathway which is difficult to study by classical methods. Thus, the development of a new detection method applicable to retroviruses is an important matter. We chose MMTV as the model system and combined the polymerase chain reaction (PCR) and in situ hybridization to develop the in situ polymerase chain reaction. On slides containing GR3A tumor sections or tissue cultures, the MMTV proviral sequences in the long terminal repeat region were first amplified with the polymerase chain reaction in situ and then detected with in situ hybridization. To localize the amplified fragments, high MW molecules were synthesized using primers tailed with complementary sequences and agarose was included in the PCR reagent mixture. MMTV proviral sequences were amplified to generate strong signals. The results demonstrated: (i) Cellular DNA can serve as the target for PCR amplification; (ii) PCR amplified fragments can be linked, through the presence of complementary primer tails, to generate large fragments that also include nonspecific DNA (primer dimers); (iii) Agarose can serve as a matrix that enhances signal localization; (iv) A target sequence of 167 bp can be detected; and (v) Cell morphology can be maintained through the PCR process. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD91-25088).
Keywords: Base Sequence DNA, Viral/*GENETICS HIV/GENETICS Mammary Tumor Viruses, Mouse/GENETICS Nucleic Acid Hybridization Polymerase Chain Reaction Proviruses/*GENETICS RNA-Directed DNA Polymerase/GENETICS RNA, Viral/GENETICS THESIS 920430
M9240932
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Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.
