Biomedical Engineering Reference
In-Depth Information
12
Molecular Analysis of Mitochondrial DNA Point
Mutations by Polymerase Chain Reaction
Lee-Jun C. Wong, Bryan R. Cobb, and Tian-Jian Chen
Summary
Mitochondrial respiratory chain disorders are clinically and genetically heteroge-
neous. There are several mitochondrial DNA (mtDNA) point mutations responsible for
common mitochondrial diseases such as mitochondrial encephalopathy, lactic acidosis,
stroke-like events, myoclonic epilepsy and ragged red fibers, neuropathy, ataxia, retini-
tis pigmentosa, and Leber's hereditary optic neuropathy. As a result of the clinical over-
lap, it is usually necessary to analyze more than one mutation for a patient suspected of
a mitochondrial disorder. Molecular diagnosis is often performed using polymerase chain
reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis of the most
likely point mutations. However, this method is time-consuming and often produces
problems associated with incomplete restriction enzyme digestion. In addition, PCR/
RFLP analysis may not be able to detect a low percentage of heteroplasmy. For a more
effective method of diagnosing mtDNA disorders, we have developed a multiplex PCR/
allele-specific oligonucleotide (ASO) dot blot hybridization method to simultaneously
analyze 11 point mutations. The PCR products from a DNA sample containing a
homoplasmic wild-type or mutant mtDNA sequence will hybridize to either the wild-
type or the mutant ASO probe. The PCR products of a heteroplasmic DNA sample will
hybridize to both wild-type and mutant ASO probes. This PCR/ASO method allows the
detection of low percentage mutant heteroplasmy.
Key Words:
Mitochondrial disorders; mtDNA mutation; heteroplasmy; oxidative
phosphorylation (OXPHOS) disease; MELAS; MERRF; NARP; LHON; mitochondrial
dysfunction; Leigh's syndrome; respiratory chain disorders.
1. Introduction
Mitochondrial respiratory chain disorders are a group of clinically and
genetically heterogeneous diseases
(
1-3
)
. Diagnosis is difficult because of the
broad spectrum of phenotypic manifestations and the considerable clinical
overlap between these disorders. The heteroplasmic nature of pathogenic point
