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A non‐radioactive protein truncation test for the sensitive detection of all stop and frameshift mutations

A non‐radioactive protein truncation test for the sensitive detection of all stop and frameshift... A new method for mutation detection is described, which is a technical advancement of the protein truncation test. The new technique is non‐radioactive and highly sensitive for detection of virtually all sequence mutations, which lead to a stop signal or to the shift of the translation frame. The method includes four steps: 1) capture of the interesting sequence copies out of the sample by binding to an immobilized complementary sequence, 2) PCR amplification of the gene fragment to be analyzed with primers coding both for amino‐ and carboxy‐terminal tags, 3) in vitro transcription and translation, and 4) analysis of the translation products by Western blot. As an evaluation of the new method, we detected mutated gene copies at a dilution of 1 to 40 compared to the non‐mutated gene. Using the method, we were able to detect a mutation in the adenomatous polyposis coli tumor suppressor gene (APC) in a stool sample of a colorectal cancer patient. This mutation could not be detected by direct sequencing of the amplified APC gene fragment. Hum Mutat 19:165–172, 2002. © 2002 Wiley‐Liss, Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Mutation Wiley

A non‐radioactive protein truncation test for the sensitive detection of all stop and frameshift mutations

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References (12)

Publisher
Wiley
Copyright
Copyright © 2002 Wiley Subscription Services, Inc., A Wiley Company
ISSN
1059-7794
eISSN
1098-1004
DOI
10.1002/humu.10024
pmid
11793475
Publisher site
See Article on Publisher Site

Abstract

A new method for mutation detection is described, which is a technical advancement of the protein truncation test. The new technique is non‐radioactive and highly sensitive for detection of virtually all sequence mutations, which lead to a stop signal or to the shift of the translation frame. The method includes four steps: 1) capture of the interesting sequence copies out of the sample by binding to an immobilized complementary sequence, 2) PCR amplification of the gene fragment to be analyzed with primers coding both for amino‐ and carboxy‐terminal tags, 3) in vitro transcription and translation, and 4) analysis of the translation products by Western blot. As an evaluation of the new method, we detected mutated gene copies at a dilution of 1 to 40 compared to the non‐mutated gene. Using the method, we were able to detect a mutation in the adenomatous polyposis coli tumor suppressor gene (APC) in a stool sample of a colorectal cancer patient. This mutation could not be detected by direct sequencing of the amplified APC gene fragment. Hum Mutat 19:165–172, 2002. © 2002 Wiley‐Liss, Inc.

Journal

Human MutationWiley

Published: Feb 1, 2002

Keywords: adenomatous polyposis coli; APC; hybrid capture; in vitro synthesis protein assay; IVSP; mutation detection; protein truncation test; PTT; tumor suppressor

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