1. Happy Monday! Checking off: Notes on Ch 20.1, 20.2 With your group, discuss what you know about these: – Methylation and Acetylation – Genetic Engineering/Biotechnology – Restriction enzymes – Gel Electrophoresis

2. Methylation and Acetylation

3. 2005-2006 The BIG Questions… How can we use our knowledge of DNA to: – diagnose disease or defect? – cure disease or defect? – change/improve organisms? What are the techniques & applications of biotechnology? – direct manipulation of genes for practical purposes

4. 2005-2006 Biotechnology Today Genetic Engineering – manipulation of DNA – if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with – this “unit” is a survey of those tools… Our tool kit…

5. 2005-2006 Bioengineering Tool kit Basic Tools – restriction enzymes – ligase – plasmids / cloning – DNA libraries / probes Advanced Tools – PCR – DNA sequencing – gel electrophoresis – Southern blotting – microarrays

6. 2005-2006 Cut, Paste, Copy, Find… Word processing metaphor… – cut restriction enzymes – paste ligase – copy plasmids – Bacteria transformation PCR – find Southern blotting / probes

7. 2005-2006 Cut DNA Restriction enzymes – restriction endonucleases – discovered in 1960s – evolved in bacteria to cut up foreign DNA (“restriction”) protection against viruses & other bacteria

8. 2005-2006 What do you notice about these phrases? radar racecar Madam I’m Adam Able was I ere I saw Elba a man, a plan, a canal, Panama Was it a bar or a bat I saw?

9. Restriction Enzymes Also known as restriction endonucleases, are enzymes that cut a DNA molecule at a particular place. They are essential tools for recombinant DNA technology. The enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to six nucleotides.

11. 2005-2006 Restriction Enzymes, c’td 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 EcoR I, Hind III, BamH I, Sma I Madam I’m Adam CTGAATTCCG GACTTAAGGC CTG|AATTCCG GACTTAA|GGC  

12. 2005-2006 AATTC GAATTC G G G G G CTTAAG GAATTC CTTAAG CTTAA CTTAAG DNA ligase joins the strands. DNA Sticky ends (complementary single-stranded DNA tails) Recombinant DNA molecule AATTC G G CTTAA How restriction enzymes are used Restriction enzyme cuts the DNA Add DNA from another source cut with same restriction enzyme

13. 2005-2006 Paste DNA Sticky ends allow: – H bonds between complementary bases to anneal Ligase – enzyme “seals” strands bonds sugar- phosphate bonds covalent bond of DNA backbone

14. Then what? DNA, cut at restriction sites, will be different lengths and will contain genes within those lengths. To analyze, mix with solutions that will allow them to travel through a medium based on size, shape, and charge. Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

15. Homework Complete Pre-lab activity for Transformation Lab – Get to Know Bacterial Transformation – Flowchart all steps