DNA extraction from formalin-fixed tissues and genetic diversity of Baiji (Lipotes vexillifer)

Archival, formalin-fixed tissue is an invaluable resource for molecular genetic study, but the utilization of nucleic acid of formalin-fixed tissue is generally problematic. Because of the negative effect of formalin on proteinase K and Taq DNA polymerase, the cross-linking between proteins and DNA, DNA degradation and other reasons, in order to successfully extract and amplify DNA from formalin-fixed tissue I formalin removal, intensive protein digestion, DNA purification, and PCR optimization should all be considered. In this study, we report an integrated and modified protocol to efficiently extract and amplify DNA from baiji ( Lipotes vexillifer)tissues, which were stored in unbuffered formalin for years. We used three pretreatment methods to remove formalin: gradual dehydration by alcohol, GTE buffer displacement and critical point drying, by comparison the DNA from samples treated by critical point drying is the best in quality and quantity. Intensive proteinase K treatment including increased proteinase K concentration and prolonged digestion time also greatly enhances quality and quantity of DNA. Because DNA from unbuffered formalin-fixed tissue was heavily degraded, a DNA target size of approximately 500bp or smaller was found to be optimal for PCR amplification, in addition nested PCR can greatly increase the positive results of amplification. As a result, DNA of 21 baiji specimens were successfully extracted and partial control region, of mitochondrial DNA were amplified and sequenced. All the specimens have identical sequences, which indicates very low genetic diversity of baiji