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Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining


Human lactate dehydrogenase (LDH) — B(H) mutant genes were analyzed by polymerase chain reaction (PCR) and DNA conformation polymorphism. We used polyacrylamide gradient gel and silver staining procedures for DCP analysis, and observed abnormal migration patterns in individuals heterozygous for the LDH-B deficiency. Subsequent sequence determination of the mutant alleles consistently resulted in detection of three single base substitutions (transversions), viz., a C to A at residue “35” (GCG, Ala→GAG, Glu), a T to G at residue “172” (TTT, Phe→GTT, Val), and an A to T at residue “176” (ATG, Met→TTG, Leu). Furthermore, mismatched PCR or amplification refractory mutation system was developed for the rapid screening and confirmation of these mutations. These amino acid replacements may cause conformational changes in neighboring residues; this probably affects the active site arrangement and results in the loss of enzyme activity.

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The residue numbers used in this manuscript are designated as in X-ray diffraction studies, and are different from the genetic codon in the LDH-B gene numbered consecutively after the translation initiation codon

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Maekawa, M., Sudo, K., Kitajima, M. et al. Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining. Hum Genet 91, 163–168 (1993).

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  • Silver Staining
  • Amino Acid Replacement
  • Mutation System
  • Single Base Substitution
  • Polyacrylamide Gradient