Biomaterial Database

Nonradioactive characterization of low-level heteroplasmic mitochondrial DNA mutations by SSCP-PCR enrichment. 1996

E J Sherratt, and A W Thomas, and J W Gagg, and J C Alcolado

University of Wales College of Medicine, UK.

Mitochondrial DNA (mtDNA) mutations have been implicated in an increasing number of human diseases. Many of these mutations are heteroplasmic and are only present at low levels in readily accessible human tissue such as blood. The technique of single-stranded conformational polymorphism (SSCP) allows the detection of mtDNA variants from peripheral blood, but characterization of these variants by automated sequencing is hampered by the low level of heteroplasmy. We have therefore developed a technique for the enrichment of mtDNA mutations that allows reliable sequence data to be obtained even if the variant mtDNA represents only 1% of the total mtDNA. The procedure involves the excision, purification and subsequent PCR amplification of selected DNA fragments from SSCP gels. The techniques can be applied to other heterogeneous mutations such as mosaic mutations in skin biopsies or somatic oncogene mutations in tumor tissue.

UI	MeSH Term	Description	Entries
D004252	DNA Mutational Analysis	Biochemical identification of mutational changes in a nucleotide sequence.	Mutational Analysis, DNA,Analysis, DNA Mutational,Analyses, DNA Mutational,DNA Mutational Analyses,Mutational Analyses, DNA
D004272	DNA, Mitochondrial	Double-stranded DNA of MITOCHONDRIA. In eukaryotes, the mitochondrial GENOME is circular and codes for ribosomal RNAs, transfer RNAs, and about 10 proteins.	Mitochondrial DNA,mtDNA
D005838	Genotype	The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS.	Genogroup,Genogroups,Genotypes
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012680	Sensitivity and Specificity	Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed)	Specificity,Sensitivity,Specificity and Sensitivity
D016133	Polymerase Chain Reaction	In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.	Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016622	Silver Staining	The use of silver, usually silver nitrate, as a reagent for producing contrast or coloration in tissue specimens.	Silver Nitrate Staining,Nitrate Staining, Silver,Staining, Silver,Staining, Silver Nitrate
D017243	MERRF Syndrome	A mitochondrial encephalomyopathy characterized clinically by a mixed seizure disorder, myoclonus, progressive ataxia, spasticity, and a mild myopathy. Dysarthria, optic atrophy, growth retardation, deafness, and dementia may also occur. This condition tends to present in childhood and to be transmitted via maternal lineage. Muscle biopsies reveal ragged-red fibers and respiratory chain enzymatic defects. (From Adams et al., Principles of Neurology, 6th ed, p986)	Fukuhara Disease,Myoclonic Epilepsy with Ragged Red Fibers,Fukuhara Syndrome,MERRF,Myoclonic Epilepsy Associated with Ragged-Red Fibers,Myoclonic Epilepsy and Ragged Red Fibers,Myoclonic Epilepsy with Ragged-Red Fibers,Myoclonus with Epilepsy with Ragged Red Fibers,Myoencephalopathy Ragged-Red Fiber Disease,Myoclonic Epilepsy Associated with Ragged Red Fibers,Myoencephalopathy Ragged Red Fiber Disease,Syndrome, Fukuhara,Syndrome, MERRF
D018807	Polymorphism, Single-Stranded Conformational	Variation in a population's DNA sequence that is detected by determining alterations in the conformation of denatured DNA fragments. Denatured DNA fragments are allowed to renature under conditions that prevent the formation of double-stranded DNA and allow secondary structure to form in single stranded fragments. These fragments are then run through polyacrylamide gels to detect variations in the secondary structure that is manifested as an alteration in migration through the gels.	SSCP,Single-Stranded Conformational Polymorphism,Conformational Polymorphism, Single-Stranded,Conformational Polymorphisms, Single-Stranded,Polymorphism, Single Stranded Conformational,Polymorphisms, Single-Stranded Conformational,Single Stranded Conformational Polymorphism,Single-Stranded Conformational Polymorphisms