Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction.
We developed a new method to amplify cell DNA in situ using the polymerase chain reaction (PCR). Proviral sequences of mouse mammary tumor virus (MMTV) contained in cultured cells and tissue sections were amplified intracellularly using a thermal cycler. Two techniques were employed to maintain the localization of the amplified DNA. First, complementary tails at the 5' ends of the oligonucleotide primers resulted in the synthesis of high molecular weight concatamers containing the target sequences. Second, the PCR was carried out in a thin film of agarose solidified over the tissue sections. The specifically amplified and localized DNA was then detected by in situ hybridization (ISH). Our results demonstrate that (a) DNA in tissue sections can serve as the target for the polymerase chain reaction in situ, (b) cell morphology is maintained, and (c) a target of 167 BP can be specifically detected in individual cells. This technique should be generally applicable to amplifying cellular DNA targets in tissue sections for detection in situ.
Chiu
K P
KP
Department of Medicine, School of Medicine, University of California, Davis 95817.
Cohen
S H
SH
Morris
D W
DW
Jordan
G W
GW
D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005347:Fibroblasts / Q000382:microbiology / Q000648:ultrastructure; D008325:Mammary Neoplasms, Experimental / Q000473:pathology* / Q000648:ultrastructure; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D012091:Repetitive Sequences, Nucleic Acid
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides