The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and...The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that展开更多
Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for ...Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for V.anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage.We characterized the library by end-sequencing 50 randomly selected clones.This generated 93 sequences with a total length of 57 485 bp covering 1.4% of the whole genome.Of these sequences,58(62.4%) were homologous to known genes,30(32.3%) were genes with hypothetical functions,and the remaining 5(5.3%) were unknown genes.We demonstrated the utility of this library by PCR screening of 10 genes.This resulted in an average of 6.2 fosmid clones per screening.This fosmid library offers a new tool for gene screening and cloning of V.anguillarum,and for comparative genomic studies among Vibrio species.展开更多
文摘The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that
基金Supported by the National Basic Research Program of China (973 Program)(No 2006CB101803)the High Technology Research and development Program of China (863 Program)(No 2006AA6100310)the National Natural Science Foundation of China (No 30871935)
文摘Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for V.anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage.We characterized the library by end-sequencing 50 randomly selected clones.This generated 93 sequences with a total length of 57 485 bp covering 1.4% of the whole genome.Of these sequences,58(62.4%) were homologous to known genes,30(32.3%) were genes with hypothetical functions,and the remaining 5(5.3%) were unknown genes.We demonstrated the utility of this library by PCR screening of 10 genes.This resulted in an average of 6.2 fosmid clones per screening.This fosmid library offers a new tool for gene screening and cloning of V.anguillarum,and for comparative genomic studies among Vibrio species.
