Restriction Mapping of Plasmid DNA

Restriction Maps Restriction enzymes can be used to construct maps of plasmid DNA Restriction enzymes can be used to construct maps of plasmid DNA Allows identification of restriction sites found in the sequence of the plasmid Allows identification of restriction sites found in the sequence of the plasmid Alternative means to discover DNA sequence without sequencing Alternative means to discover DNA sequence without sequencing Can be used to determine which restriction enzymes to use for recombinant techniques Can be used to determine which restriction enzymes to use for recombinant techniques

Getting the Information Isolate the DNA Isolate the DNA Digest it with restriction enzymes Digest it with restriction enzymes Breaks DNA into various size fragments Breaks DNA into various size fragments Sort fragments by size using gel electrophoresis Sort fragments by size using gel electrophoresis Can sort DNA fragments differing by only a single nucleotide in length Can sort DNA fragments differing by only a single nucleotide in length Fragment lengths can be used to assemble map of relative positions Fragment lengths can be used to assemble map of relative positions

Digestion Can do single or multiple digestions Can do single or multiple digestions Single digestion consists of only one enzyme in the mixture Single digestion consists of only one enzyme in the mixture Multiple digestions include many or all the enzymes desired Multiple digestions include many or all the enzymes desired Step-wise process gives the most information Step-wise process gives the most information Digest with individual enzymes and include a multiple digestion Digest with individual enzymes and include a multiple digestion Use different sizes of the fragments to construct the map Use different sizes of the fragments to construct the map

Making the Map The gel will be stained and photographed after running The gel will be stained and photographed after running Distances migrated by DNA fragments will be measured from the photograph of the gel Distances migrated by DNA fragments will be measured from the photograph of the gel A standard curve will be produced by plotting distances migrated by ladder fragments against the size of the fragments A standard curve will be produced by plotting distances migrated by ladder fragments against the size of the fragments Semilog graph paper must be used to obtain a linear plot Semilog graph paper must be used to obtain a linear plot The standard curve will be use to determine fragment sizes of the digested plasmid DNA The standard curve will be use to determine fragment sizes of the digested plasmid DNA A circular map will be constructed from the data A circular map will be constructed from the data