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Title: Screening of point mutations by multiple SSCP analysis in the dystrophin gene

Abstract

Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimalmore » for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.« less

Authors:
; ;  [1]
  1. Hospital Sant Pau, Barcelona (Spain)
Publication Date:
OSTI Identifier:
134754
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1492
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; 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; PATIENTS; HEREDITARY DISEASES; MORTALITY; MUSCLES; GENES; GENE MUTATIONS; MUTATION FREQUENCY; SIZE; TRANSCRIPTION; GENETIC MAPPING; DETECTION; HUMAN X CHROMOSOME; PROTEINS; QUANTITATIVE CHEMICAL ANALYSIS; EFFICIENCY; MESSENGER-RNA; ELECTROPHORESIS

Citation Formats

Lasa, A., Baiget, M., and Gallano, P. Screening of point mutations by multiple SSCP analysis in the dystrophin gene. United States: N. p., 1994. Web.
Lasa, A., Baiget, M., & Gallano, P. Screening of point mutations by multiple SSCP analysis in the dystrophin gene. United States.
Lasa, A., Baiget, M., and Gallano, P. Thu . "Screening of point mutations by multiple SSCP analysis in the dystrophin gene". United States. doi:.
@article{osti_134754,
title = {Screening of point mutations by multiple SSCP analysis in the dystrophin gene},
author = {Lasa, A. and Baiget, M. and Gallano, P.},
abstractNote = {Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.},
doi = {},
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}
}
  • The APC gene is a putative human tumor-suppressor gene responsible for adenomatous polyposis coli (APC), an inherited, autosomal dominant predisposition to colon cancer. It is also implicated in the development of sporadic colorectal tumors. The characterization of APC gene mutations in APC patients is clinically important because DNA-based tests can be applied for presymptomatic diagnosis once a specific mutation has been identified in a family. Moreover, the identification of the spectrum of APC gene mutations in patients is of great interest in the study of the biological properties of the APC gene product. The authors analyzed the entire coding regionmore » of the APC gene by the PCR-single-strand conformation polymorphism method in 42 unrelated Italian APC patients. Mutations were found in 12 cases. These consist of small (5-14 bp) base-pair deletions leading to frameshifts; all are localized within exon 15. Two of these deletions, a 5-bp deletion at position 3183-3187 and a 5-bp deletion at position 3926-3930, are present in 3/42 and 7/42 cases of the series, respectively, indicating the presence of mutational hot spots at these two sites. 17 refs., 2 figs., 1 tab.« less
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  • Mutations in the p53 tumor-suppressor gene are commonly found in human cancers of diverse origin. Once of a number of methods developed to analyze large numbers of DNA samples for specific mutations is the single-strand conformation polymorphism (SSCP) analysis. This method is particularly well suited for analysis of tissues, such as brain tumors, with mixed cell populations. It takes advantage of the fact that, in a mixed cell population containing DNA with and without a mutation (e.g., the p53 gene mutation), both molecular species will be amplified by the PCR. A mutation within a PCR-amplified DNA fragment will alter themore » secondary structure of the amplified fragment and affect its electrophoretic mobility in a nondenaturing gel. The DNA fragments with the mutation are detected as an aberrantly migrating allele that can be seen concurrently with the wild-type allele. Although many studies have used this technique to screen for p53 mutations in tumors, with detection of a number of different mutations the limit of detection of point mutations in a background of wild-type DNA is not known. To test this, mixtures of mutant DNA from tumor D317 with a G-to-A point mutation in codon 272 of the p53 gene; or from tumor D263 (with a G-to-A point mutation in codon 175 of the p53 gene) and wild-type DNA from leukocytes, in ratios of 1:100, 5:95, 10:90, 15:85, 50:50, and 30:70, were prepared. The mixtures containing 100 ng of DNA were amplified using standard PCR technique. After the double-stranded DNAs were denatured, the DNA samples were loaded and electrophoresed on a nondenaturing acrylamide gel. The mutant allele was detectable even when the ratio of mutant to wild-type DNA was 5:95 in tumor D317. For tumor D263, the mutant allele was detectable when the ratio of mutant to wild-type DNA was 15:85, and it was not detectable at 10:90.« less
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