Excision Repair:

1. glycosylase removes the damaged nitrogenous base from the DNA by breaking the
bond between the base and the deoxyribose
2. AP endonuclease creates a nick at the apurinic site.
3. An exonuclease expands the nick creating a single stranded regions in the DNA
4. DNA polymerase synthesizes new DNA
5. Ligase seals the nick
TRANSLATION INITIATION PRO
1. The small ribosomal subunit positions itself correctly by interacting with (binding)
the Shine-Delgarno sequence within the mRNA. *1*
2. This places the start codon in the P site of the small subunit. *2*
3. A charged fmet-tRNA binds the start codon. *3*
4. The large subunit assembles in such a way that the initiator tRNA is in the
completed P site. *4*
5. A charged tRNA that decodes the second codon enters the A site of the ribosome.
*5*
6. Peptidyl transferase catalyzes the formation of a peptide bond between the amino
acid in the P site and the A site of the ribosome. *6*
7. The ribosome translocates three nucleotides down the mRNA. *7*
8. The uncharged tRNA is now in the E site, the tRNA carrying a dipeptide is in the P
site and the A site is empty awaiting another charged tRNA. *8*
TRANSLATION INITIATION EUK
1. Second, the mRNA must be transported from the nucleus to the cytoplasm.
2. The small riboosomal subunit scans the mRNA from the 5' methylated Cap until it
locates the start codon. The start codon is almost always the first AUG in the mRNA.
3. This places the start codon in the P site of the small subunit.
4. A charged met-tRNA binds the start codon.
5. The large subunit assembles in such a way that the initiator tRNA is in the completed
P site.
6. A charged tRNA that decodes the second codon enters the A site of the ribosome.
7. Peptidyl transferase catalyzes the formation of a peptide bond between the amino
acid in the P site and the A site of the ribosome.
8. The ribosome translocates three nucleotides down the mRNA.
9. The uncharged tRNA is now in the E site, the tRNA carrying a dipeptide is in the P
site and the A site is empty awaiting another charged tRNA.
CLONING by COMPLEMENTATION
1. Identify a mutation that causes the bacterial to be sensitive to weak acid.
2. Show that the mutation is recessive. You also discover that the mutant does
not grow on solid media (petri dish) that has low pH. The wild type grow well
on solid media that has low pH
3. Make a genomic library from a wild type bacterial strain. The vector is a
Plasmid

4. Transform the plasmid into the mutant strain

5. Plate the cell on media that has a low pH
6. Isolate any colonies that grow

Cdna library
1. purify mRNA from cell
2. add oligo dt primer
3. Add reverse transcriptase
4. Denature cDNA-mRNA ds nucleic acid
5. 2nd strand cDNA synthesis occurs
6. Add S1 nuclease to cut hairpin loop
7. Ligate into vector
AMPLIFICATION
96 to denature 52 allow primers to anneal to template 72 max DNA synthesis