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Title: Identification of APC mutations and evaluation of their expression level using a functional screening assay

Abstract

A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

Authors:
; ;  [1]
  1. Istituto Nazionale Ricerca sul Cancro, Genova (Italy); and others
Publication Date:
OSTI Identifier:
133396
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-0124
Resource Type:
Journal Article
Journal Name:
American Journal of Human Genetics
Additional Journal Information:
Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENES; DNA SEQUENCING; GENE MUTATIONS; POLYPEPTIDES; BIOLOGICAL MARKERS; MESSENGER-RNA; GENETICS; DNA-CLONING; POLYMERASE CHAIN REACTION; ABSORPTION SPECTROSCOPY

Citation Formats

Varesco, L, Gismondi, V, and Bafico, A. Identification of APC mutations and evaluation of their expression level using a functional screening assay. United States: N. p., 1994. Web.
Varesco, L, Gismondi, V, & Bafico, A. Identification of APC mutations and evaluation of their expression level using a functional screening assay. United States.
Varesco, L, Gismondi, V, and Bafico, A. 1994. "Identification of APC mutations and evaluation of their expression level using a functional screening assay". United States.
@article{osti_133396,
title = {Identification of APC mutations and evaluation of their expression level using a functional screening assay},
author = {Varesco, L and Gismondi, V and Bafico, A},
abstractNote = {A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.},
doi = {},
url = {https://www.osti.gov/biblio/133396}, journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {Thu Sep 01 00:00:00 EDT 1994},
month = {Thu Sep 01 00:00:00 EDT 1994}
}