In-situ polymerase chain reaction: foundation of the technology and today's options.
"In situ PCR" is the marriage of two established technologies in molecular genetics, the polymerase chain reaction (PCR) and in situ hybridization (ISH). It is based on the amplification within intact cells or tissue sections of specific gene sequences, or mRNA species, to levels detectable by ISH and/or immunohistochemistry. Methods to achieve in situ PCR, while sharing fundamental steps, have differed between different laboratories. On the basis of our own experience, in situ PCR appears to be best suited for the detection of DNA in single cell preparations, in which fixation and pre-treatments can be optimally controlled. Emphasis is placed on the requirement for appropriate and meaningful controls at the multiple steps involved. It is instructive to the view the emergence of this new technology in perspective. In situ PCR has not developed in isolation and is just one of several creative approaches that have been employed in recent years to study nucleic acids (DNA and RNA) intracellularly. Some approaches are more suitable for detection of mRNA, or viral RNA, while others are more easily applied to chromosomal DNA. Some further techniques, such as the isothermal self-sustained sequence replication (3SR), refined in-situ transcription (PRINS), or high sensitivity histochemical detection systems, will complement or even add to the potential of situ PCR. It is highly probable that tests will emerge, based on investigation of unique genetic markers, with important roles in specialized diagnostic laboratories for the evaluation of viral diseases, as well as hematological and other malignancies.
Long
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Department of Medicine, Tufts New England Medical Center Hospitals, Boston, MA 02111, USA. aidan/.long@es.nemc.org
D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*