인체 대장암 및 자궁경부암에서 PCR-SSCP법을 이용한 Ki-ras 암유전자의 점돌연변이에 관한 연구 = A Study on the Detection of c-Ki-ras2 Point Mutations in Human Colorectal and Cervical Cancer by Single Strand Conformation Polymorphism Analysis of Polymerase Chain Reaction Products

국문 초록 (Abstract)

인체 대장암 20례와 자궁경부암 10례의 암조직을 대상으로 c-Ki-ras 유전자 codon 12와 13에서의 점돌연변이를 알아보고자 polymerase chain reaction(PCR)과 single-strand conformation polymorphism(SSCP) 검사법을 시행하였다. 대장암의 경우 20례중 9례(45%)에서, 자궁경부암의 경우 10례중 1례(10%)에서 양성으로 나왔다. 대장암에서는 codon 12에서 GGT가 TGT로의 치환이 4례로서 가장 많았으며, AGT, CGT로의 치환이 각 1례였고 codon 13에서는 GGC가 GAC로의 치환이 2례, TGC로의 치환이 1례인 것으로 나타났다. 자궁경부암의 경우 codon 12에서 GGT가 AGT로의 치환이 1례인 것으로 나타났다.

다국어 초록 (Multilingual Abstract)

In an attempt to clarify the role of genetic alteration in the genesis of human colorectal and cervical cancers, tissue specimens from 20 patients with colorectal cancer and 10 patients with cervical cancer were examined for the presence of point mutation in K-ras2 exon 1 by single strand conformation polymorphism analysis of PCR product.
Exon I of c-Ki-ras2 was amplified by polymerase chain reaction(PCR) and comparison was made between the normal and mutated genes by nondenaturing polyacrylamide gel electrophoresis(PAGE) of PCR product and nucleotide sequence analysis using asymmetric PCR with direct sequencing.
Genomic DNA from white blood cells were used as normal control and those from A427 cell line were used as mutated control.
1.PCR product from A427 cell line showed a distinct migration shift pattern compared to the normal control in PAGE and the direct sequencing indicated that nucleotide sequence of codon 12 was mutated from GGT to GAT. In addition, there was a loss of normal allele in A427 cell line.
2.PCR product from 9 cases(45%) out of a 20 colon cancer patients showed migration shifts in PAGE, and all of these 9 patients invariably demonstrated mutation of ras gene, either in codon 12(6 cases:30%) or 13(3 cases:15%). The base substitutions in codon 12 were: from GGT to AGT(1), CGT(1), or TGT(4).
Base changes in codon 13 were from GGC to TGC(1) or GAC(2).
3.PCR product from 1 cases(10%) out of a 10 cervical cancer patients showed migration shrifts in PAGE, Invariably demonstrated mutation of ras gene in codon 12. The base substitutions was from GGT to AGT(1)
TQE incidence of point mutation of c-Ki-ras in colon cancer was high, however, it was low in cervical cancer, which showed the relation of ruts gene mutation with colon cancer.
PCR -SSCP analysis is a simple, rapid and efficient method of detection of point mutation, especially when dealing with multiple samples.

목차 (Table of Contents)

• Ⅰ.서론
• Ⅱ.재료 및 방법
• Ⅲ.결과
• A.인체 종양 세포주에서 c-Ki-ras2에 대한 PCR-SSCP 분석
• B.인체 대장암 및 자궁경부암에서 c-Ki-ras2에 대한 PCR-SSCP 분석 결과
• Ⅳ.고찰
• Ⅴ.맺는말