Résumé
Objective To establish a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method which can identify deer blood. Methods We analyzed DNA sequences of cytochrome b (cytb) from different species (i.e. sika deer, red deer, pig, ox, sheep, and duck) and selected two DNA restriction enzymes of EcoRV and MseI to distinguish deer from other species and to sika deer from red deer, respectively. Genomic DNA of every sample was extracted by blood genomic DNA extraction kit. After PCR of cytb fragment, digestion by EcoRV or MseI was conducted and the results were analyzed. The capacity of this method to identify different proportional blood mix from different species also was determined. Results Using the PCR-RFLP method, EcoRV digestion made two fragments of 287 bp and 185 bp in deer, but no digestion in other species; MesI digestion made two fragments of 281 bp and 191 bp in sika deer, but three fragments of 281 bp, 126 bp, and 65 bp in red deer. The 191 bp fragment was a characteristic marker of sika deer, and the 126 bp was for red deer. The minimum detected proportion added to blood of deer with other sample was 3%, 6% of sika deer with red deer. Conclusion A PCR-RFLP method according to the sequences of cytb gene is established. This method can identify the blood of sika deer rapidly and conveniently.
Résumé
It is known that no restriction endonuclease cleavage site exists at 12 codon in N-ras gene and at 12 and 13 codon in K-ras gene.Amismatched base was incorporated at the 3' end primer to creat a kind of restriction endonuclease cleavage site.Then it was possible for us to analyze the point mutation of the above mentioned codons with the polymerase chain reaction-restriction fragment length polymorphism.In this paper,the study of the point mutation at 12 and 61 codon in c-Ha-ras,at 12 codon in N-ras,and at 12 and 13 codon in K-ras was reported.