Conservative

*Semi Conservative*  Winner

Dispersive

The Most Beautiful Experiment in all of Biology

1. label old DNA with heavy Nitrogen ¹⁵N

2. give cells natural lighter Nitrogen ¹⁴N

3. DNA then are separated by by density gradient centrifugation, to determine whether new DNA ends up in the same molecules as old DNA or if new DNA ends up as molecules separate from old DNA.

(note: they invented CsCl density gradient to carry out experiment)

Autoradiography

1. cultured E. Coli Cells in the presence of ³H-Thymidine

2. gently extracted chromosomes were cover with photographic gel

3. Radioactivity from ³H allowed visualization of entire chromosome

4. gave a glimpse at replication intermediates θ structure

the single site of DNA replication initiation

(the bubble)

1. Crude E. Coli exctract + ¹⁴C thymidine + ATP

2. Purify DNA and check for ¹⁴C

(figured out that dNTP's are essential precursors for DNA synthesis

later typically used ³²P-labeled dNTP's for incorporation assays

γ gamma β beta α alpha phosphates

which one remains in bond?

1. Exonuclease _______________.

2. Endonuclease________________.

3. Klenow fragment ______________.

1. is a type of enzyme that can initiate DNA replication in areas where there is some sort of nick.

2. is a type of enzyme that can initiate DNA replication anywhere.

3. Domain 2 and 3 of pol I

1. obtain pure DNA sample

2. lightly nick it with DNAse

3. then treat with radioactive dNTPs and pol I

You now have DNA with radioactive nucleotides incorporated

1. domain 2 of pol I catches a mistake

2. kicks the mistake off and pol I backs up

3. replication continues

Surfer Dude!

Polymerase Chain Reaction (PCR)

1. use oligonucleotide with known gene of interest

2. denature with heat in the presence of many synthetic primers that hybridize with gene of interest

3. decrease temperature to allow hybridization and add Klenow (or Taq) and dNTPs to stimulate primer directed polymerization

(repeat steps over and over)

exponential amplification of gene of interest

DNA polymerase that allowed PCR to become the fully mature technology that it is today because it requires such high temperatures to denature it.

Gene of interest

temperature sensitive, a protein has a single amino acid mutation that causes it to be functional at low temperature, but not at high temperatures.

Out of all the Ts mutants were defective none were defective in pol I activity, suggesting that pol I is not critically important for DNA replication

1. mutated non-functional pol I did not slow DNA replication

2. mutated non-functional pol II did not slow DNA replication

3. mutated non-functional pol III did slow down DNA replication

Pol III core

1. what are the different divisions?

2. which are bigger?

3. what are their functions?

1. α,ε,θ

2. big, medium, small

3. polymerase 5'-3', proofreading exnuclease 3'-5', no function

Pol I

Domain 1

Domain 2

Domain 3

1. chews stuff up

2. read 3' to 5' and catches mistakes

3. polymerase corrects mistakes