Rapid progress in the molecular technology has stimulated attempts to establish genetic diagnosis of human disease. Practical use of DNA amplification method proved to be very useful in genetical analysis. Polymerase chain reaction (PCR) is one of the most popular method for DNA amplification technology. There are some point mutations which can be detected by the restriction endonuclease digestion of amplified DNA. Such technology is generally called as PCR-RFLP (restriction fragment length polymorphism). However, some mutations do not effect on recognition sites of any restriction endonucleases. In those cases, a new primer can be designed to create any recognition sites. The primer and PCR using the primer are called as mismatched primer and mismatched PCR, respectively. Before and after restriction endonuclease digestion of amplified DNA, they are electrophoresed on agarose gel or polyacrylamide gel to distinguish the cleaved bands. It is important to confirm that the amplified DNAs do only contain the target DNA, without non-specific DNAs. Electrophoretic methods are useful for this purpose. In this experimental protocol we describe the procedures for PCR techniques, restriction endonuclease digestion, and mismatched PCR. Also we describe some examples using these procedures to detect genetic mutations in lactate dehydrogenase and serum cholinesterase genes.

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The allantoin level in human serum was measured by capillary electrophoresis (CE). CE was carried using a fused-silica capillary tube (57cm long, 75μm I.D.). Analytical conditions were pH 9.0 50mM Na₂B₄O₇-HCl as a running buffer, at a voltage of 25kV, with detection set at 214nm. The correlation factor was 0.999 in the range of 2-400μM. The coefficients of variation for the measurement of allantoin were lower than 10%. The recovery was 92-113%. The allantoin concentration was not increased for 24 hours in storage time at 4°C and room storage temperature, but was markedly increased in storage under light conditions for 24 hours. Allantoin was not detected in healthy subjects except one case. However, in cases of rheumatism, allantoin levels were significantly higher than in healthy subjects. Capillary electrophoresis is useful to measure the allantoin concentration in human serum.

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A method for an ultrasensitive silver-based color staining of proteins described by Sammons et al. was applied to the detection of oligonucleotides on polyacrylamide gels with slight modifications in the method of fixation. With these modifications, we can stain even tetranucleotide bands such as dA₄. Furthermore, the sensitivity of staining of Hae III fragments of φX 174 RF DNA is several times higher than with ethydium bromide staining. The sensitivity of this silver-staining differs markedly among various homo-oligonucleotides. The limit of detection of rA₁₀ or dA₁₀ is approximately 40pg per mm² of cross-sectional area. Homo-oligonucleotides, dG₁₀ and dC₁₀, are also detectable. However, the dT₁₀ band concentrated more than 1, 000-fold could not be detected on the polyacrylamide gel by silver staining. Thus, the silver staining method is very useful for the sensitive detection of oligonucleotides as short as tetranucleotides that are not stained by ethidium bromide.

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Determination of oligoclonal IgG bands (OGB) in cerebrospinal fluid (CSF) has diagnostic value in multiple sclerosis. In this paper, we describe a modified method for the detection of OGB in unconcentrated CSF. We used agarose electrophoresis followed by protein transfer to PVDF membrane and immunodetection with the following two modifications. First, we omitted equilibration step of the gels with transfer buffer before blotting. Second, for enhanced staining of alkaline phosphatase, we used a suitable mixture of BCIP and NBT for developing solution. These modifications made this method approximately 30 times more sensitive than immunofixation method. Of 83 unconcentrated CSF samples examined, 11 were positive for OGB with our method. On the other hand, with the immunofixation, despite using twenty-five times concentrated samples, only four were positive and four were equivocal. These results suggest that our modified method is useful for the screening of OGB in unconcentrated CSF.

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Abnormal IgA molecule, which lacked in disulfide bridges between α chains, was detected from the sera of patients of monoclonal IgA gammopathy with albuminuria by the method of urea-SDS-PAGE performed in the reducing-agent-free condition. Throughout the procedure, protein in the specimen was kept away from any reducing agent to avoid an artificial production of half molecules during the experiment. A band of 80kDa protein, which showed immunoreactivity to both anti-α- and anti-λ-chain antibodies, was detected in sera from four patients of monoclonal IgA gammopathy accompanied with albuminuria. The molecular mass and the immunoreactivity indicated that the 80kDa protein was so-called“IgA half molecule”(α₁λ₁). The band of α₁λ₁ molecule was faint on the gel in the specimens from patients of no albuminuria. The abnormal α₁λ₁ molecule was eluted from the column of gel permeation chromatography into fractions corresponding to 320kDa when it was performed using urea-free buffer. The chromatographic behavior suggested the non-covalent interaction between the abnormal α₁λ₁ molecules in the non-denaturing buffer condition.

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We have developed a latex agglutination immunoassay method for free light chains of immunoglobulins in urine and clarified its clinical significance. Bence Jones protein, which was qualitatively detected in urine with the traditional immunoelectrophoresis, could be quantitatively determined by this method. The free light chain determination was useful for diagnosis and prognosis of multiple myeloma that is associated with monoclonal free light chains. The increase in monoclonal free light chains in urine was found in patients with primary macroglobulinemia, primary amyloidosis, and systemic lupus erythematosus. These results suggest that free light chains in urine are important markers for multiple myeloma and related diseases in clinical practice.

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The phospholipase D (PLase D) was purified from crude extract of leaves of cabbage to electrophoretically homogeneity by acetone precipitation, Octyl-Sepharose CL-4B column hydrophobic chromatography and Mono-Q column chromatography. The molecular weight of purified PLase D was estimated to be approximately 87kDa by SDS-PAGE. The N-terminal amino acid sequences of PLase D preparation and derivative peptides of PLase D prepared by limited proteolysis with Staphylococcus aureus V8 Protease and Achromobacter Lysyl Endopeptidase were determined. These sequences were compared with amino acid sequence deduced from cDNA of rice, maize and castor bean PLase D. As a result, it was clarified that the primary structure of PLase D from cabbage was similar to that from other plants. The N-terminal amino acid sequence analysis was revealed that the band of PLase D preparation, demonstrated as single band on SDS-PAGE, contained three proteins with slightly different N-terminal residues.

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The age-depended levels of creatine kinase (CK) isoenzymes in 544 juvenile outpatients and 37 healthy adults were evaluated by CK isoenzyme analysis with cellulose acetate membrane electrophoresis. From the results, the age-depended levels of CK-MM activities were relatively low in the tested all subjects. Further, higher activities of CK-BB and CK-MB were depended on younger juveniles. In contrast, mitochondrial CK (m-CK) was detected in 511 (93.9%) of the tested 544 juveniles and in all of the tested 37 healthy adults. In addition, higher levels of m-CK were found in the tested all age groups, compared with those levels of CK-BB and CK-MB.

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