Cloning and identification of a specific standard DNA fingerprint fragment from Chinese herbal medicine Fritillaria cirrhosa D. Don / 中国药学杂志

ABSTRACT

OBJECTIVE: To establish a standard method for preserving the Fritillaria cirrhosa D. Don DNA fingerprint fragments. METHODS: Independently developed F. cirrhosa test kit was used to extract genomic DNA. Specific F. cirrhosa DNA fragments were cloned in vitro, and ligated into a carrier vector then transformed into a self replicating host cells to produce a large amount of specific F. Cirrhosa DNA. Plasmid DNA was extracted using Plasmid Extraction Kit and confirmed by PCR or restriction digestion of the insert. Bacterial colonies carrying authentic Sichuan F. cirrhosa specific DNA sequences were stored at -80℃. Stability was monitored at intervals of 1, 2, 3 and 6 months after DNA recovery using the boiling method of genomic DNA extraction. RESULTS: The amplified F. cirrhosa DNA clones showed clear bands in the electrophoresis, and had stable results in the repeated verification test. The amplified clones from resuscitated bacterial colonies which had been stored for 1, 2, 3, 6 months at -80℃ still displayed bright and clear bands after electrophoresis. CONCLUSION: It is feasible and effective to preserve DNA fingerprint of F. Cirrhosa by the established method, and this simple and reliable method can be used as the basis of establishing the new genes database of traditional Chinese medicine.