Ch3 Recombinant DNA

 

Mixing and matching of DNA molecules to make any combination you want

 

restriction endonucleases

            - bacterial proteins

            - cut dsDNA (break phosphodiester bonds) at specific sequences

            - Table 9.1

         if they are displaced, creates sticky ends

o       can be either 5’ overhang or 3’ overhang

         if not displaced, creates blunt ends

            - the sequence recognized by the enzyme is the recognition sequence

         can be a 4bp sequence, a 6bp sequence, or 8bp sequence (or more for some exotic enzymes)

         often recognition seq are palindromic

         frequency of sites in DNA is

-          for 4bp rec. seq. 1/4X1/4X1/4X1/4 = 1/256

-          for 6bp 46 = 1/4096

-          for 8bp 48 = 1/65,500bp

           

 Agarose gel electrophoresis

 

Recombinant DNA

- cut two different DNAs from different sources with an enzyme which leaves sticky ends

- mix two together and use DNA ligase to form phosphodiester bonds between two different DNA fragments

            - make a recombinant DNA molecule (Fig. 9.7)

            - vectors – specialize DNA molecule that

         has recognition sequences for some enzymes

o       can put DNA from another source in there, the bigger the better

         has an origin of replication and so can be replicated in a cell

         has a selectable marker such as an antibiotic resistance gene

         Table 9.2 shows several kinds

o       vary in host, carrying capacity

o       plasmids are common vectors

         extrachromosomal circular double stranded DNA

         cell can live without, but benefit host under certain conditions

         antibiotic resistance

         pathogenicity (such as Shigella toxin)

 

Molecular cloning fragments of DNA

 

- pUC19

            - more advanced vector

            - amp selectable marker

            - ori

            - multiple cloning site (many unique restriction sites in small amount of sequence)

            - can cut with one enzyme and insert

            - can use two enzymes and have directional cloning