Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells.
Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.
Christian
A T
AT
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA. christian4@llnl.gov
Pattee
M S
MS
Attix
C M
CM
Reed
B E
BE
Sorensen
K J
KJ
Tucker
J D
JD
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics; D005090:Exodeoxyribonucleases; D005784:Gene Amplification; D015870:Gene Expression; D016158:Genes, p53; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics*
D015342:DNA Probes; D004270:DNA, Circular; D012333:RNA, Messenger; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes