Establishment Of Mosquito AFLP System

Objective To investigate the key factors of Amplified Fragment Length Polymorphism(AFLP) system of mosquito, it contains the critical protocols of the preparation with template DNA,restriction endonuclease digestion and ligation of adapters,pre-amplification and selective amplification. Then AFLP system is established, and optimized further. At least, we also filt selective AFLP primers optimized for the advance study of AFLP system with mosquito.Method Culex pipiens pallens is the objective of study, Phenol-chloroformed isolated mosquito genome DNA is detected by DU730for concentration and purity, And designed primers using mitochondria CoiA genome, which is validated through PCR amplification,purification sequencing and Blast; DNA digestion incubate in37℃water at different times which separates1h,2h,3h,4h, and run out in a2.0%agarose gel; ligation of adapters in16℃water bath respective for2h,3h,overnight; The ligation products of three groups are diluted5times,10times.20times, which are participating in the pre-amplification and then run out in a2.0%agarose gel; The pre-amplification products diluted5times,10times,20times participate in the selective amplification. what's more, the second cycles of selective amplified program is modified for system optimized, and run out in a2.0%agarose gel. The AFLP system optimized run out again with8forward primers of EcoRI and50reverse primers of Msel, and run out in a2.0%agarose gel. Following, the selective primers group which have a clear, complete and detachable bands in the selective amplification are choosed. Fluorescent labeled forward primers, the selective amplification with the selective primers group run again and carry out capillary electrophoresis. So the selective primers group are filted again for further study of amplified fragment length polymorphism system of mosquito.Result Genome DNA isolated with mosquito have quite good quality, the ratio of its OD260/OD280approach to1.8and its concentration generally be greater than400μg/ml, Homology of PCR amplified fragment aligned by GenBank is at a range of99%-100%; The best incubation time of DNA digestion is3h in37℃water, which have good diffusible bands; ligation of adapters in16℃have poor correlation of bath time; The desirable template of pre-amplification is ligation products diluted5 times; Pre-amplified products and the second cycles of25,the selective amplification could produce clear, complete and detachable bands, the selective primers group are filted by2.0%agarose gel and CE, whose minimal detachable bands could be more than ten strips and whose maxima detachable bands could be about one handred and fifty stripsConclusion AFLP system is established and optimized and the selective primers group are filted provide stronger foundation for further research about mosquito that contains Genetic diversity,he construction of genetic fingerprints and gene localization.