In situ strand displacement amplification: An improved technique for the detection of low copy nucleic acids

Strand displacement amplification (SDA) is an isothermal, enzymatic method for the exponential synthesis of target nucleic acids which traditionally r equires two pairs of oligoprimers. One pair, described as "bumpers", serves to generate target specific single stranded DNA after heat denaturation of double stranded DNA subsequent to primer annealing and extension. We repor t here the in situ (IS) application of SDA (IS-SDA) using nicked DNA target s which eliminate the need for heat denaturation and bumpers. productive ni cking can be generated by restriction endonuclease activity, exposure to dr y heat, paraffin embedding, or mild depurination. IS-SDA, followed by in si tu hybridization (ISH) allows for the routine detection of single copy HIV- 1 DNA as well as I-IPV DNA in both cell lines and tissue sections. Detectio n of hepatitis C RNA in liver biopsy specimens using reverse transcription IS-SDA (RT-IS-SDA) was performed with direct incorporation of the reporter nucleotide. The amplification of RNA targets does require bumper primers. a lthough not an initial denaturation step. Extensive analyzes demonstrated t hat SDA-ISH is as sensitive as polymerase chain reaction (PCR)-ISH and that each are more sensitive than in situ hybridization coupled to catalyzed si gnal amplification (CSA). Moreover, SDA-ISH is technically much simpler tha n PCR-ISH because it is isothermal and does not require an initial denatura tion.