Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ 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.

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High Throughput Cellular Localization of Specific Plant mRNAs by Liquid-Phase in Situ Reverse Transcription-Polymerase Chain Reaction of Tissue Sections

PLANT PHYSIOLOGY ◽

10.1104/pp.123.4.1203 ◽

2000 ◽

Vol 123 (4) ◽

pp. 1203-1212 ◽

Cited By ~ 46

Author(s):

Hinanit Koltai ◽

David McKenzie Bird

Keyword(s):

Polymerase Chain Reaction ◽

Liquid Phase ◽

High Throughput ◽

Reverse Transcription ◽

Cellular Localization ◽

Chain Reaction ◽

Tissue Sections ◽

Polymerase Chain

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Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections

Histochemistry ◽

10.1007/bf00571876 ◽

1993 ◽

Vol 99 (2) ◽

pp. 151-162 ◽

Cited By ~ 108

Author(s):

A. A. Long ◽

P. Komminoth ◽

E. Lee ◽

H. J. Wolfe

Keyword(s):

Polymerase Chain Reaction ◽

Chain Reaction ◽

Tissue Sections ◽

Polymerase Chain

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Polymerase chain reaction amplification of three different Trypanosoma cruzi DNA sequences from human chagasic cardiac tissue.

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.1998.59.563 ◽

1998 ◽

Vol 59 (4) ◽

pp. 563-570 ◽

Cited By ~ 32

Author(s):

D Olivares-Villagómez ◽

T L McCurley ◽

C L Vnencak-Jones ◽

C E Carter ◽

R Correa-Oliveira ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Trypanosoma Cruzi ◽

Dna Sequences ◽

Cardiac Tissue ◽

Polymerase Chain Reaction Amplification ◽

Chain Reaction ◽

Reaction Amplification ◽

Polymerase Chain

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High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166944 ◽

1996 ◽

Vol 54 ◽

pp. 896-897

Author(s):

G. W. Hacker ◽

I. Zehbe ◽

J. Hainfeld ◽

A.-H. Graf ◽

C. Hauser-Kronberger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Messenger Rna ◽

High Performance ◽

Detection System ◽

Direct Methods ◽

Genetic Alterations ◽

Chain Reaction ◽

Protein Encoding ◽

Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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