Related Experiment Videos
A restriction endonuclease assay for expression of human alpha-amylase isozymes
Shigeru Hokari1, Kae Miura, Iwao Koyama
1Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama-machi, Iruma, Saitama 350-0495, Japan. shhokar@saitama-med.ac.jp
Background:
The alpha-amylase isozymes can be detected separately by electrophoresis; however, sometimes the identification is difficult because of their microheterogeneity. In the present study, we tried to establish a convenient method for the detection of alpha-amylase isozyme expression.
Methods:
The procedure is based on three different restriction sites presented in those genes; a PstI site in both AMY 2A and 2B genes, a HaeII site in both AMY 1 and 2A genes, and a BamHI site in AMY 2B gene. After amplification from total tissue RNAs by RT-PCR with primers that were able to cover each exon, the products were cleaved with corresponding restriction endonucleases.
Results:
This method was applied to human samples from the parotid gland, liver (non-hepatoma), hepatoma and white blood cells (WBCs). The results indicated that the parotid gland and hepatoma (also liver) clearly expressed AMY 1 and AMY 2B genes, respectively. However, AMY 2B gene was also expressed apparently in WBCs, which produced salivary-type isozyme of the alpha-amylase, although the amylase protein was not able to identify for the hepatic isozyme.
Conclusions:
The method presented here might be convenient and useful for the determination of alpha-amylase isozyme expression in humans.