The important info of DNA is…
…in the center (between the antiparallels)
In DNA replication
we first replicate one strand and use it as a template to create a new one
We can make old DNA:
- radioactive
- heavier
Atomic mass of regular N
14
Meselson & Stahl
Tested the hypothesis of DNA replication**
Atomic mass of heavy N
15
Meselson & Stahl’s Test
- Culture bacteria in 15N medium (heavier nitrogen)
- They changed bacteria to 14 N medium (regular)
Enzyme that is in charge of DNA replication
DNA polymerase
Arthur Kornberg
studied DNA polymerase and DNA replication***
PPP
is a reactive group**
DNA helicase
opens up the helix in ADN during replication - unzips, unwinds
Primase
puts in primers and adds them to adn**
Ligase
this enzyme joins little pieces of replicated DNA together
Topoisomerase
untangle 2 topoisomers of DNA during replication
wherever they meet, it makes a cut (breaks bonds) and separates them
With DNA polymerase
1/1000 chances of getting wrong :(
With Proofreading
1/1000000 chances of getting wrong :(
Mismatch detection
You now have enzymes that scan through DNA and detect where is a wrong nucleotide inserted
With mismatch detection
1/(10^9) :D (only 3 mutations)
There are _______ bases in our DNA
3 billion
Kornberg’s polymerase
20 to 50 nucleotides/sec vs 1000 nucleotides/sec :(
Mutant DNA Polymerase***
If Kornberg’s polymerase I is not the main DNA polymerase, the you could find a mutant with effective DNA polymerase
Grifith’s experiment
Mice infected with bacteria
S strain=virulent
Tricks of cloning
Partial Digest
Partial digest
Adding less restriction enzyme(ej EcoRI) in order to get a partial representation of the gene.
