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Titolo:
A methylation PCR approach for detection of fragile X syndrome
Autore:
Panagopoulos, I; Lassen, C; Kristoffersson, U; Aman, P;
Indirizzi:
Univ Lund Hosp, Dept Clin Genet, S-22185 Lund, Sweden Univ Lund Hosp Lund Sweden S-22185 Dept Clin Genet, S-22185 Lund, Sweden
Titolo Testata:
HUMAN MUTATION
fascicolo: 1, volume: 14, anno: 1999,
pagine: 71 - 79
SICI:
1059-7794(1999)14:1<71:AMPAFD>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
POLYMERASE CHAIN-REACTION; SODIUM BISULFITE; DNA DIAGNOSIS; CGG REPEAT; AMPLIFICATION; INSTABILITY; CYTOSINE; URACIL; DERIVATIVES; MUTATION;
Keywords:
bisulfite treatment; CGG repeats; diagnostic test; direct visualization; ethidium bromide; fragile X; methylation; PCR;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
32
Recensione:
Indirizzi per estratti:
Indirizzo: Panagopoulos, I Univ Lund Hosp, Dept Clin Genet, S-22185 Lund, Sweden UnivLund Hosp Lund Sweden S-22185 S-22185 Lund, Sweden
Citazione:
I. Panagopoulos et al., "A methylation PCR approach for detection of fragile X syndrome", HUM MUTAT, 14(1), 1999, pp. 71-79

Abstract

Fragile X syndrome is associated with the expansion of the number of CGG trinucleotide tandem repeats at the 5' untranslated region of the FMR1 gene. The number of CGG trinucleotide repeats in normal individuals ranges between 5 and 50, in asymptomatic carrier individuals it ranges between 50 and 200, and in affected individuals it is more than 200 CGG repeats. In addition, in affected individuals the cytosine residues in the CGG repeats and theadjacent CpG island are methylated and the FMR I gene is transcriptionallyinactive. The most common diagnostic method for the detection of the syndrome is Southern blot analysis. Methods based on the polymerase chain reaction (PCR) could facilitate the rapid screening of large numbers of individuals by accurately determining the number of CGG repeats. Current PCR techniques for amplification of CGG repeats are, however, inefficient and unreliable because of their 100% C+G composition. Thus, most of the described PCR protocols require subsequent Southern blot analysis and autoradiography. We present a novel PCR approach for the diagnosis of fragile X syndrome based on the methylation sensitive conversion of C residues to U by bisulfite on single-strand DNA and subsequent amplification of the antisense strand withspecific primers. A PCR with primers for methylated C residues will amplify the CpG dinucleotide region upstream to CGG repeats exclusively in affected males. As a result of extensive mismatch between primers and bisulfite treated DNA, no PCR fragments will be obtained in normal and transmitting males, Moreover, the bisulfite treat ment dramatically reduces the C+G component of the region; thus, the high T-m and the strong secondary structures are no longer obstacles for PCR amplification. In normal and carrier individuals, UUG repeats (previously 3'-CCG-5') in the antisense strand can easilybe amplified and visualized on a gel by ethidium bromide staining, We applied our method on 25 males previously diagnosed by Southern blot analysis. All the samples were easily and accurately diagnosed. The method has considerable advantages compared with other diagnostic tests for fragile X syndrome. Hum Mutat 14:71-79, 1999. (C) 1999 Wiley-Liss, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/03/20 alle ore 15:40:45