1. 3. DNA Ligase & 4. Gel Electrophoresis

2. Yesterday... Introduced restriction enzymes and methylases Restriction enzymes are enzymes that cut DS DNA at a specific region or recognition site – Two examples EcoRI SmaI 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ 5’ – G G G C C C – 3’ 3’ – C C C G G G – 5’

3. Yesterday... The original function of RE’s is to protect the host cell from invading viral DNA RE’s will find recognition site on foreign DNA – Break the phosphodiester linkages between specific bases Methylases are enzymes that protect the host DNA from attacking itself – Methylases add a –CH3 group to a nucleotide in the recognition site – Preventing RE’s from attacking own DNA – We can use this to prevent RE’s from attacking our gene

4. Today 3. DNA Ligase Adding nucleotides back together Opposite of REs 4. Gel Electrophoresis DNA separation

5. 3. DNA Ligase From DNA replication, we know that DNA Ligase is used to connect Okazaki fragments. Question: What is DNA Ligase actually doing to reconnect the DNA fragments? Answer:

6. 3. DNA Ligase Uses a condensation rxn – Thus, WATER IS REMOVED Opposite of hydrolysis – A phosphodiester linkage is made T4 DNA Ligase – special ligase used for blunt ends A A T T C – 3’ G – 5’ 5’ – G 3’ – C T T A A 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’

7. 3. DNA Ligase IMPORTANT NOTE DNA LIGASE will not reform bonds between fragments cut by different RE’s If your gene has a sticky end from EcoRI, it will only bond to ANOTHER sticky end made by EcoRI – Reforms A-G phosphodiester linkage A A T T C – 3’ G – 5’

8. 4. GEL ELECTROPHORESIS Biotechnology technique that separates DNA fragments based on: – CHARGE – SIZE Uses the physical and chemical properties of DNA to separate **Don’t forget......DNA is negative (due to O - groups on Phosphate)

9. 4. GEL ELECTROPHORESIS Nucleotides (A, T, G, C) have a fairly similar molar mass Thus, all nucleotides have a similar mass-to- charge ratio THUS, the main difference between DNA fragments is – Length (# of bases in the fragment) 5’ – A G G T T T A C G C A – 3’ 5’ – G G C C A T – 3’

10. 4. GEL ELECTROPHORESIS So what do we use to actually separate the gel?

11. 4. GEL ELECTROPHORESIS So what do we use to actually separate the gel? Gel – square/rectangular slab that consists of agarose (polysaccharide found in seaweed) – Contains ‘wells’ at one end where DNA is loaded – DNA is mixed with dye and glycerol Glycerol is heavy, so the DNA will sink to bottom of gel Buffer sol’n contains electrolytes to help electrical current travel

12. 4. GEL ELECTROPHORESIS So we turn on the power supply and what ACTUALLY happens? DNA is separated by size and charge Size: -Gel starts and smaller DNA fragments travel faster -Can move through pores in the gel easier

13. 4. GEL ELECTROPHORESIS So we turn on the power supply and what ACTUALLY happens? DNA is separated by size and charge Charge: -–ve charge is placed in the end near the wells -+ve charge is placed at opposite end -DNA will be repelled from –ve end -DNA will move toward +ve end

14. 4. GEL ELECTROPHORESIS Final product:

15. 4. GEL ELECTROPHORESIS Final product: DNA fragments are visible by staining the gel with ETHIDIUM BROMIDE – Fluoresces under UV light – **inserts between nucleotides The DNA fragments can be excised out of gel for further use