Section J-克隆DNA的分析与应用PPT.ppt

cDNA clones have defined organization. A run of A residues defines the clone’s 3’-end. There will be a stop codon at its upstream. If the clone is complete, there also will be a start condon. These two codon indicates an ORF. J4-1 Organiztion J4 Organization of cloned genes The presence and polarity of any gene in a genomic clone is not obvious (5’ and 3’ end) It can be determined by mapping and probing experiments To determine: which genomic sequences are present in the mature mRNA transcript The absent sequences are usually introns and sequences upstream of the transcription start site and down stream of the 3’-processing site. Start and stop sites for transcription regulatory sequences. J4 Organization of cloned genes J4-2 Mapping cDNA on genomic DNA The genomic clone is digested on a gel and then subjected to Southern blot using all or part of the cDNA as a probe. Using full length cDNA as probe can show which genomic restriction fragments contain sequences also present in the cDNA Using a probe from one end of a cDNA can show the polarity of the gene in the genomic clone. Some of the restriction sites will be common in both clones but may be different distances apart. These can often help to determine the organization of the genomic clone. J4 Organization of cloned genes J4-3 S1 nuclease mapping determines the precise 5’- and 3’- ends of RNA transcripts. Sequence ladder is required to determine the precise position S1 nuclease is an enzyme which specifically hydrolyses single-stranded RNA or DNA. RNA 5’ DNA 3’ * 5’ 3’ RNA 5’ DNA 3’ 5’ 3’ PAGE Analysis Add S1 nuclease J4 Organization of cloned genes J4-4 Primer extension Determine the 5’ ends of RNA molecules using reverse transcriptase to extend an antisense DNA primer in the 5’ to 3’ direction. Sequence ladder is required to determine the precise position J4 Organization of cloned genes RNase T1: cleaves after G RNase U2: after A RNase Phy M: after A and U Bacillus cereus RNase: after U and C J