Nonradioactive Characterization of Low-Level Heteroplasmic Mitochondrial DNA Mutations by SSCP-PCR Enrichment
نویسندگان
چکیده
منابع مشابه
Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations.
Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injec...
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The presence of hundreds of copies of mitochondrial DNA (mtDNA) in each human cell poses a challenge for the complete characterization of mtDNA genomes by conventional sequencing technologies. Here we describe digital sequencing of mtDNA genomes with the use of massively parallel sequencing-bysynthesis approaches. Although the mtDNA of human cells is considered to be homogeneous, we found wides...
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Single-strand conformation polymorphism (SSCP) is a powerful technique used to identify mutations in short pieces of DNA (1). In this technique, the DNA of interest is often amplified by the polymerase chain reaction (PCR) and then denatured by heat or alkali treatment before electrophoresis on a non-denaturing polyacrylamide gel. Differences in mobility of either of the single strands, compare...
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Single-strand conformation polymorphism (SSCP) has become the most widely used method to screen new mutations or already-established polymorphisms. Several protocols have been described to overcome the two main problems related to this technique, which are separation of both strands and detection. For the former, there are methods using a strong denaturant such as methylmercury hydroxide to enh...
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Mitochondria contain their own DNA (mtDNA), which codes for 13 proteins (all subunits of the respiratory chain complexes), 22 tRNAs and 2 rRNAs. Several mtDNA point mutations as well as deletions have been shown to be causative in well-defined mitochondrial disorders. A mixture of mutated and wild type mtDNA (heteroplasmy) is found in most of these disorders. Inheritance of mtDNA is maternal, a...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 1996
ISSN: 0736-6205,1940-9818
DOI: 10.2144/19962003430