Methodologies for the detection of nucleotide substitutions as well as small insertions and deletions in clinical molecular diagnosis of human genetic diseases have been recently reviewed.[1] For unknown mutations, although the gold standard is direct DNA sequencing, it is usually not practical to routinely sequence the entire coding regions of candidate gene or genes in patients suspected of having the genetic disease. Very often, a high-throughput, low-cost mutation screening method is used to quickly scan the coding exons and intron/exon junctions for the presence of mutations, followed by sequencing of suspected regions prior to sequencing of the regions containing the mutations. Commonly used screening methods for unknown mutations include denaturing gradient gel electrophoresis (DGGE),[2] temperature gradient gel electrophoresis (TGGE),[3] single-strand conformation polymorphism (SSCP),[4] heteroduplex analysis (HA),[5,6] chemical mismatch cleavage (CMC),[7] enzyme mismatch cleavage (EMC),[8,9] protein truncation test (PTT),[10] mismatch binding protein (MutS),[11] and cleavase fragment length polymorphism (CFLP).[12] The drawbacks of these methods include low sensitivity (SSCP, HA), difficulty in casting denaturing gradient gel (DGGE), synthesis of long GC clamped primers (DGGE, TGGE), the inability to detect homozygous mutations (HA, CMC, EMC), high background (CMC, EMC), lack of documented reports (CFLP, EMC, MutS), and preferential elimination of unstable mutant transcripts (PTT).[1] Furthermore, most of these methods are used for the detection of mutations in nuclear genes. Application of these methods to the detection of mitochondrial DNA (mtDNA) mutations is limited because of the heteroplasmic feature of pathogenic mtDNA mutations and numerous benign, homoplasmic single nucleotide polymorphisms. Thus a unique requirement for the molecular analysis of mtDNA mutations is the ability to detect heteroplasmic mtDNA mutations and to distinguish them from homoplasmic sequence varia tions. In search of an effective and sensitive mutation detection method, we recently developed the temporal temperature gradient gel electrophoresis (TTGE) that not only resolves the wild-type and mutant homoduplexes and the wild-type/mutant heteroduplexes into four distinct bands, but also detects a heteroplasmic or heterozygous change in the background of homoplasmic or homozygous polymorphism. Here we describe the utility of TTGE in the detection of mutations in both nuclear and mitochon-drial genomes.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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