In situ hybridization, in situ transcription, and in situ polymerase chainreaction

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the pri nciples of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA- RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridiz ation of a labeled probe to a complementary target sequence, whereas in sit u transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the ca se of in situ PCR (ISPCR), it is the repeated in situ duplication of both t he sense and antisense strands of DNA to increase the number of copies of t he target sequence. ISH, IST, and ISPCR each have their advantages and disa dvantages. The purpose of this chapter is to address in situ considerations requried of these techniques, emphasizing tissue fixation, pre-hybridizati on steps, DNA probes, RNA probes, oligoprobes, and probe labeling. Five suc cessfully used protocols are presented as examples. Any given nucleotide ta rget sequence may have its own unique set of optimum conditions, thus requi ring some adjustment in the hands of the user.