Quickie Intro to DNA Technologies 2006-2007

1953 article in Nature Watson and Crick

Double helix structure of DNA

Cut DNA Restriction enzymes restriction endonucleases discovered in 1960s evolved in bacteria to cut up foreign DNA “restriction” protection against viruses & other bacteria bacteria protect their own DNA chemically & by not using the base sequences recognized by the enzymes in their own DNA

Discovery of restriction enzymes Werner Arber Daniel Nathans Hamilton O. Smith Restriction enzymes are named for the organism they come from: EcoR1 = 1st restriction enzyme found in E. coli Werner Arber discovered restriction enzymes. He postulated that these enzymes bind to DNA at specific sites containing recurring structural elements made up of specific basepair sequences. Hamilton Smith verified Arber's hypothesis with a purified bacterial restriction enzyme and showed that this enzyme cuts DNA in the middle of a specific symmetrical sequence. Other restriction enzymes have similar properties, but different enzymes recognize different sequences. Ham Smith now works at Celera Genomics, the company who sequenced the human genome. Dan Nathans pioneered the application of restriction enzymes to genetics. He demonstrated their use for the construction of genetic maps and developed and applied new methodology involving restriction enzymes to solve various problems in genetics. Restriction enzyme movie

Restriction enzymes Madam I’m Adam Action of enzyme cut DNA at specific sequences restriction site symmetrical “palindrome” produces protruding ends sticky ends Many different enzymes named after organism they are found in EcoR1, HindIII, BamH1, Sma1 CTGAATTCCG GACTTAAGGC  CTG|AATTCCG GACTTAA|GGC 

Biotech use of restriction enzymes GAATTC CTTAAG DNA Restriction enzyme cuts the DNA Sticky ends (complementary single-stranded DNA tails) AATTC G AATTC G G CTTAA G CTTAA Add DNA from another source cut with same restriction enzyme AATTC G G AATTC CTTAA G DNA ligase joins the strands. Recombinant DNA molecule GAATTC CTTAAG

Paste DNA Sticky ends allow: Ligase H bonds between complementary bases to anneal Ligase enzyme “seals” strands bonds sugar-phosphate bonds covalent bond of DNA backbone

Gel Electrophoresis Separation of DNA fragments by size DNA is negatively charged moves toward + charge in electrical field agarose gel “swimming through Jello” smaller fragments move faster cut DNA with restriction enzymes

Gel Electrophoresis

Gel Electrophoresis

Measuring fragment size compare bands to a known “standard” usually lambda phage virus cut with HindIII nice range of sizes with a distinct pattern

And from that comes a host of… Biotech Tools