1997 Fiscal Year Final Research Report Summary
Assessment of genetic changes in the carcinogenesis of squamous cell lung cancer using in situ PCR.
| Project/Area Number |
07457286
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Thoracic surgery
|
|---|
| Research Institution | Tohoku University |
|---|
Principal Investigator |
SATO Masami Tohoku University, Institute of Development, Aging and Cancer, Instructor, 加齢医学研究所, 助手 (30250830)
|
|---|
| Project Period (FY) |
1995 – 1997
|
| Keywords | in situ PCR / K-ras / point mutation / PCR / in situ hybridization / direct in situ PCR / indirect in situ PCR |
|---|
| Research Abstract |
In situ PCR is a new technique for the localization of low copy number sequences. We investigated to establish 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 postfixation 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 seme preparation confirmed desired amplification.We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations. We also applied direct in situ PCR,but this method failed to detect the point mutation. Huge non-specific incorporation of digoxigenin to several length of DNA fragments caused low specificity of this technique.
|
