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

• electrophoresis - separating charged molecules in an electric field

• agarose is like Jello - comes as a powder, mix in buffer, heat, pour in mold and it cools

• make so has wells at one end, load in DNA

• apply electric field, gel moves through gel

• big pieces move slow, small ones move fast

• stain with something that makes nucleic acid be visible

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

• step 1 – make recombinant DNA molecules with vectors
• step 2 – make host cell take up recombinant DNA molecules (transformation)
  • CaCl2
  • electroporation
  • selectable markers come in handy here to select those few bacteria that take up the plasmid
  • each colony on a plate comes from a single cell taking up a single plasmid

- 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