分子生物学课件7.DNA tech案例实例.ppt

NNG GATCCNN NNCCTAG GNN internal fragment cut with Bam HI (6-base cutter) remove internal fragment “left arm” “right arm” cut with Sau 3A (4-base cutter) which has ends compatible with Bam HI: NNN GATCNNN NNNCTAG NNN isolate ~20 kb fragments human genomic DNA (isolated from many cells) Bam HI sites: combine and treat with DNA ligase “left arm” “right arm” “left arm” “right arm” package into bacteriophage and infect E. coli 1 2 3 4 5 6 genomic library of human DNA fragments in which each phage contains a different human DNA sequence 7 isolation of ~20 kb fragments provides optimally sized DNAs for cloning in bacteriophage partial digestion with a frequent-cutter (4-base cutter) allows production of overlapping fragments, since not every site is cut overlapping fragments insures that all sequences in the genome are cloned overlapping fragments allows larger physical maps to be constructed as contiguous chromosomal regions (contigs) are put together from the sequence data number of clones needed to fully represent the human genome (3 X 109 bp) assuming ~20 kb fragments theoretical minimum = ~150,000 99% probability that every sequence is represented = ~800,000 Partial restriction enzyme digestion allows cloning of overlapping fragments a “contig” All possible sites: Results of a partial digestion: = uncut = cut 基因文库的构建 载体DNA的分离 真核生物DNA的分离 部分酶解 片段回收(9-23 kb) 载体与插入片段的连接 包装 文库的效价测定 文库的扩增 文库的保存 重组克隆鉴定 Construction of a cDNA library reverse transcriptase makes a DNA copy of an RNA The life cycle of a retrovirus depends on reverse transcriptase retrovirus 1. virus enters cell and looses envelope 2. the capsid is uncoated, releasing genomic RNA and reverse transcriptase 3. reverse transcriptase makes a DNA copy 4. then copies the DNA strand to make it double-stranded DNA, removing the RNA with RNase H 5. the DNA is then integrated into the host cell genome w