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In-situ PCR (CAT#: STEM-MB-0206-WXH)

Introduction

In-situ means “in the original place”. In the biology context, this means in the cell, body or tissue. In-situ PCR is a technique where PCR is carried out in a section of tissue within a cell. The tissue sections of interest are attached on a microscopic slide and incubated with all necessary reagents for PCR.
DNA present inside the cells provide the template for amplification, thus this technique is useful for finding the location of a certain gene and give information on which cells in which tissue it is present




Principle

The principle of this method involves tissue fixing (to preserve the cell morphology) and subsequent treatment with proteolytic digestion (to provide access for the PCR reagents to the target DNA). The target sequences are amplified by those reagents and then detected by standard immunocytochemical protocols.

Applications

Detection of infectious disease agents including HIV-1, HBV, HPV, HHV-6, CMV, and EBV.

Procedure

1.Sample preparation
2.In-situ first strand synthesis
3.In-situ PCR using either labelled or unlabelled probes
4.In-situ Hybridization
5.Signal detection

Materials

The primers used in in-situ PCR are coupled with “marker” molecules. (e.g. biotin or digoxigenin molecule). After PCR, the amplicons can be identified using antibodies against these markers. The antibodies can be made visible by attaching a fluorescent or colorimetric enzyme to it.
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