DESCRIPTION: (Applicant's Description) The tumor suppressor genes represent an important class of genes which are implicated in the initiation and promotion of cancer. It has been observed that truncating mutations constitute the majority of disease associated mutations in this class of genes. A mutation detection strategy that specifically identifies truncating mutations, the protein truncation test (PTT), is currently accepted as the assay of choice for screening for the presence of this type of mutation. Unfortunately, PTT has drawbacks which limit its use. The principal technical objections to PTT are that it is labor intensive, time consuming, expensive, not amenable to automation, requires the use of radioisotopes, and typically requires technical personnel at the doctoral level. Of serious concern are issues of sensitivity and specificity. False negative results may result when truncation mutations are very near the ends of the mRNA. Further, PTT is incapable of detecting missense mutations, a sizable minority of tumor suppressor gene mutations. High background levels render analysis in tissue samples, which virtually always contain substantial numbers of non-malignant cells, extremely difficult. Further, these high background levels require confirmation of any positive results by sequence analysis, adding to complexity and cost. We propose to develop practical assay systems for the detection of mutations in tumor suppressor genes. The test systems should exhibit high sensitivity and specificity, be user friendly, be automatable, be immediately transferable to clinical laboratories, and be cost effective. For maximum efficiency and flexibility, we will pursue a two phase approach to the development of improved PTT assays. The first phase will utilize incorporation of fluorescently labeled amino acids into the peptide translation product and detection using capillary electrophoresis (CE). This strategy will eliminate the need for radioactivity and introduce an automated platform. Further, this strategy will allow the detection of nonconservative missense mutations using capillary isoelectric focusing techniques. Required for this approach will be the development of synthetic capability for the preparation of tRNA molecules aminoacylated with fluorescently labeled amino acids. The second phase will develop fluorescently labeled suppressor tRNA (supp-tRNA) constructs capable of directly labeling stop codons due to either nonsense or frameshift mutations. The full-length peptides, fluorescently labeled at the stop codon(s) will be detected by fluorescence spectroscopy after capture on a solid phase in a 96 well microtiter tray format. This phase of the project will build on synthetic capabilities gained during phase one.
NIH ExPorter
NIH ExPorter