Localized in situ amplification (LISA): a novel approach to in situ PCR.
Amplification of specific gene target sequences has become a routine molecular procedure in a variety of laboratories. When coupled with either a direct or indirect method of detecting amplified product, in situ amplification offers an extremely powerful investigative tool. We describe a protocol for a localized in situ amplification (LISA) reaction that includes tissue-culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections. Digoxigenin-11-dUTP was added to the amplification reaction and thus incorporated into the amplified products, providing a mechanism by which direct nonisotopic detection could be performed. To demonstrate the approach, LISA was performed on known positive Pneumocystis carinii rat lung tissues, with primers specific for the P. carinii rRNA gene sequence.
Tsongalis
G J
GJ
Department of Hospital Laboratories and Pathology, University of North Carolina, Chapel Hill 27514.
McPhail
A H
AH
Lodge-Rigal
R D
RD
Chapman
J F
JF
Silverman
L M
LM
D000818:Animals; D001483:Base Sequence; D004587:Electrophoresis, Agar Gel; D017403:In Situ Hybridization / Q000379:methods*; D008168:Lung / Q000382:microbiology; D008969:Molecular Sequence Data; D011010:Pneumocystis / Q000235:genetics; D016720:Pneumocystis Infections / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012331:RNA, Fungal / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D051381:Rats
D012331:RNA, Fungal; D012335:RNA, Ribosomal