Recombinant DNA technology – combining genes from different sources into a single molecule. The result is a transgenic organism Bacteria, like E coli, readily host recombined DNA Transgenic food crops are modified for pest resistance and increased nutrition

Plasmids – extrachromosomal DNA - are used to customize bacteria

Bacterial plasmids are copied during binary fission so they function well as vectors for inserting/copying gene DNA

Restriction enzymes ‘cut and paste’ DNA • Used by bacteria to cut up foreign DNA • Each recognizes a specific DNA sequence • Many are pallindromes Blunt cuts Staggered cuts

Restriction enzymes (endonucleases) that produce staggered cuts are used for recombining DNA The staggered cuts create ‘sticky ends’ that easily base pair

Using the same restriction enzyme on DNA from multiple sources produces complementary sticky ends Ligase brings together the sugars & phosphates of the two nucleic acids http://www.slic2.wsu.edu:82/hurlbert/micro101/images/LigaseAnimation6.gif

restriction enzyme, the plasmid and gene base-pair Cut with the same restriction enzyme, the plasmid and gene base-pair to make recombinant DNA Application: producing gene products, i.e. HGH http://www.abpischools.org.uk/res/coResourceImport/modules/hormones/en-flash/geneticeng.cfm

Recombinant DNA overview

Cloning genes in plasmids Recombinant plasmid Bacterium takes up plasmid Bacteria are allowed to divide forming a colony of clones Test each colony with antibiotics

Look for the colonies that took up the target gene(s)

GFP in Aequorea victoria

http://labs.medicine.ucsf.edu/chrislau/GFP.html

How to identify the clones with the gene of interest? • pair the gene with another for antibiotic resistance; test each clone group using antibiotics • If the gene of interest produces a protein, like HGH, see which clones can • Use a nucleic acid probe – create the base sequence that complements the inserted gene. Tag it with florescence or radioactivity. Mix with heated DNA to get visual confirmation