Rapid and Non-radioactive Method for Detection and Cloning of Rare cDNA Fragments

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Isolation and characterization of rare and desirable cDNA fragments are very important steps in biomedical research. In the present study, we developed a rapid method for non-radioactive detection and cloning of rare cDNA fragments. In order to analyze DNA molecules with high sensitivity, DNA fragments are separated on the conventional polyacrylamide gel and visualized with silver staining instead of isotopic labeling. More than 100pg of DNA were detectable on the silver-stained gel. For further characterization, we examined recovery conditions of DNA fragments isolated from the silver-stained gel. Comparative analyses of recovery conditions showed that mild stop reaction of silver staining without acetic acid and removal of silver ion from the DNA extract by precipitation with NaI, are the key steps for efficient re-amplification of extracted DNA fragments. In order to evaluate this improved technique, gene expression of Xenopus embryos was examined using non-radioactive differential display method. Isolation and characterization of the lithium-induced cDNA bands from the silver-stained gel resulted in cloning of a novel gene with partial similarity to mouse blastocyst-derived cDNA clone. In addition, whole-mount in situ hybridization showed that lithium ion induces a temporal expression of the gene. The present results successfully showed that such rapid and non-radioactive method is a potentially useful technique for basic research in various biomedical fields.
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Rapid and Non-radioactive Method for Detection and Cloning of Rare cDNA Fragments

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