Fast and efficient mutation detection method using multiplex PCR and cycle sequencing--application to haemophilia B.

J M Costa; P Ernault; D Vidaud; M Vidaud; D Meyer; J M Lavergne

Home/Archive/PubMed

PubMed · 10739381

Fast and efficient mutation detection method using multiplex PCR and cycle sequencing--application to haemophilia B.

J M Costa·P Ernault·D Vidaud·M Vidaud·D Meyer·J M Lavergne

View original source ↗Export metadata ↓

Abstract

A method using multiplex PCR followed by cycle-sequencing has been developed to detect mutations in the FIX gene. The procedure was evaluated in 45 severe or mild haemophilia B patients from 45 unrelated families. At least one deleterious mutation was identified in every haemophiliac demonstrating the efficiency of the method. Furthermore the described procedure offers many advantages compared to other screening detection methods: it is fast (less than 48 h), simple (partly automated) and of relatively low cost (it requires only one PCR).

Explore related subjects

Codon, Nonsense DNA Mutational Analysis Factor IX Frameshift Mutation Gene Deletion Genetic Testing Hemophilia B Humans Mutation, Missense Point Mutation Polymerase Chain Reaction Polymorphism, Genetic Journal Article

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

J M Costa, P Ernault, D Vidaud, M Vidaud, D Meyer, J M Lavergne. 2000. Fast and efficient mutation detection method using multiplex PCR and cycle sequencing--application to haemophilia B.. https://pubmed.ncbi.nlm.nih.gov/10739381/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2003

Nonsense-mediated decay breaks the circle?

This Commentary discusses new data from the laboratory of Matthias Hentze (Schell et al., in this issue) that begins to dissect the RNA-protein and protein-protein interactions important for the process of nonsense-mediated decay (NMD). Schell and co-workers have developed a novel tool for analysis of such interactions in vivo. Using a proteomics approach, they have identified two different protein-RNA complexes that contain human Upf (up-frameshift) proteins central to the NMD process. Intriguingly, they identified a poly[A]-binding protein associated with these complexes.

Codon, Nonsense↗

PubMed2003

Glutamine is incorporated at the nonsense codons UAG and UAA in a suppressor-free Escherichia coli strain.

Readthrough of the nonsense codons UAG, UAA, and UGA is seen in Escherichia coli strains lacking tRNA suppressors. Earlier results indicate that UGA is miscoded by tRNA(Trp). It has also been shown that tRNA(Tyr) and tRNA(Gln) are involved in UAG and UAA decoding in several eukaryotic viruses as well as in yeast. Here we have investigated which amino acid(s) is inserted in response to the nonsense codons UAG and UAA in E. coli. To do this, the stop codon in question was introduced into the staphylococcal protein A gene. Protein A binds to IgG, which facilitates purification of the readthrough product. We have shown that the stop codons UAG and UAA direct insertion of glutamine, indicating that tRNA(Gln) can read the two codons. We have also confirmed that tryptophan is inserted in response to UGA, suggesting that it is read by tRNA(Trp).

Codon, Nonsense↗

PubMed2003

Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt -1) in the NDUFS4 gene in Leigh syndrome.

Complex I deficiency, the most common cause of mitochondrial disorders, accounts for a variety of clinical symptoms and its genetic heterogeneity makes identification of the disease genes particularly tedious. Indeed, most of the 43 complex I subunits are encoded by nuclear genes, only seven of them being mitochondrially encoded. In order to offer urgent prenatal diagnosis, we have studied an inbred/multiplex family with complex I deficiency by using microsatellite DNA markers flanking the putative disease loci. Microsatellite DNA markers have allowed us to exclude the NDUFS7, NDUFS8, NDUFV1 and NDUFS1 genes and to find homozygosity at the NDUFS4 locus. Direct sequencing has led to identification of a homozygous splice acceptor site mutation in intron 1 of the NDUFS4 gene (IVS1nt -1, G-->A); this was not found in chorion villi of the ongoing pregnancy. We suggest that genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families helps to identify the disease-causing mutation. More generally, we suggest giving consideration to a more systematic microsatellite analysis of putative disease loci for identification of disease genes in inbred/multiplex families affected with genetically heterogeneous conditions.

Codon, Nonsense↗