Detection of K-ras point mutation by in situ PCR in cell suspensions: comparison of the indirect and direct methods.
In situ PCR is a new technique for the localization of low copy number sequences. We report here a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR. Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and post-fixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated: TGT) and NCI-H460 (wild type: GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the same preparation confirmed desired amplification. We also applied direct in situ PCR, but this method failed to detect the point mutation. We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations.
Sagawa
M
M
Biomarkers and Prevention Research Branch, National Cancer Institute, Rockville, MD 20850, USA.
Jones
J E
JE
Ebina
M
M
Jensen
S
S
Linnoila
R I
RI
D015139:Blotting, Southern; D002478:Cells, Cultured; D017931:DNA Primers; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers