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Application of the MutEx/ACP-PCR Method of Genotypic Selection to the Detection of K-ras Mutations

Investigators
McKinzie, Page
Institutions
DHHS/FDA - National Center for Toxicological Research
Objective
The Division has several projects in which this new technology is being developed. The specific goals of this project are to establish assays that can provide mechanistic data for chemical risk assessment and aid in establishing the relevance of rodent models for predicting human risk. The proposed research approach is to apply a recently developed method, MutEx/ACB-PCR to the detection of human and rodent k-ras GGT-GAT and GGT-GTT mutations. The assays will then be used to study the chemical induction of these mutations.

FY 2000 Accomplishments:

  1. Cloning of human k-ras wild type.
  2. Mutagenesis of human k-ras from GGT to GAT and GGT to GTT.
  3. Cloning of rat wild-type k-ras.
FY 2001 Plans:
  1. Mutagenesis of rat k-ras to make GGT to GAT and GGT to GTT mutants.
  2. Finish optimization of human and rat ACB-PCR.
  3. Optimize MutEx method for human and rat k-ras studies.
  4. Begin validation of method with unknown samples.
More information
The majority of methods for quantitating mutation require the use of selective drugs which allow mutants to grow and prevent normal cells from growing. All of these techniques require extensive cell culture and can be time-consuming and expensive. There are techniques that utilize genotypic selection that allow for a molecular amplification of the rare mutant DNA sequences and thus provide for a direct measurement of mutant frequencies.
Funding Source
Nat'l. Center For Toxicological Research
Project number
E0706601
Categories
Sanitation and Quality Standards
Risk Assessment, Management, and Communication