Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Alice Kamal Abd El Aleem, Ingolf Böhm, Samia Temtamy, Mostafa El-Awady, Mohamed Awadalla, Jörg Schmidtke, Manfred Stuhrmann

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)5 and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.

Original languageEnglish
Pages (from-to)577-584
Number of pages8
JournalHuman Genetics
Volume96
Issue number5
DOIs
Publication statusPublished - Nov 1995
Externally publishedYes

Fingerprint

Fragile X Syndrome
Digoxigenin
Southern Blotting
Polymerase Chain Reaction
Mutation
Alleles
Trinucleotide Repeats
Oligonucleotide Probes
Agar Gel Electrophoresis
Acrylamide
Nylons
Luminescence
Prenatal Diagnosis
Intellectual Disability
Sepharose
Molecular Biology
Anti-Idiotypic Antibodies
Fetus
Gels
Phosphates

ASJC Scopus subject areas

  • Genetics(clinical)
  • Genetics

Cite this

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection. / Kamal Abd El Aleem, Alice; Böhm, Ingolf; Temtamy, Samia; El-Awady, Mostafa; Awadalla, Mohamed; Schmidtke, Jörg; Stuhrmann, Manfred.

In: Human Genetics, Vol. 96, No. 5, 11.1995, p. 577-584.

Research output: Contribution to journalArticle

Kamal Abd El Aleem, Alice ; Böhm, Ingolf ; Temtamy, Samia ; El-Awady, Mostafa ; Awadalla, Mohamed ; Schmidtke, Jörg ; Stuhrmann, Manfred. / Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection. In: Human Genetics. 1995 ; Vol. 96, No. 5. pp. 577-584.
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AU - Stuhrmann, Manfred

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