Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

Abstract

Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described.

• Authors: Sha Tang, Michelle C Halberg, Kristen C Floyd, Jing Wang
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 837 Pg. 259-79 ( 2012) ISSN: 1940-6029 [Electronic] United States
• PMID: 22215554 (Publication Type: Journal Article)

Chemical References

• DNA, Mitochondrial
• Oligodeoxyribonucleotides
• DNA Restriction Enzymes

Topics

• Alleles
• Autoradiography
• Blotting, Southern (methods)
• DNA Mutational Analysis (methods)
• DNA Restriction Enzymes (metabolism)
• DNA, Mitochondrial (genetics, isolation & purification, metabolism)
• Electrophoresis, Agar Gel
• Humans
• Mitochondrial Diseases (genetics)
• Nucleic Acid Hybridization (methods)
• Oligodeoxyribonucleotides (genetics, metabolism)
• Point Mutation
• Polymerase Chain Reaction