A restriction enzyme cuts at specific sequences. However, if there is a point mutation, the site that would normally be cut by the restriction enzyme is not.

Mechanism of action of restriction enzymes

1. bind in major groove of DNA
2. migrate along double helix until it binds to its desired sequence
3. this will put a kink in the structure of the enzyme
4. this will activate the phosphodiester bonds within DNA so they are easier to break

• it has a helix turn helix motif
• what this allows is for two desired palendrome sequences to be opposed
• this will allow the same AA side chain to interact with one strand of the DNA of inerest and the opposite chain can bind to the opposite strand perfectly

Name the difference between a blunt end and sticky end

• blunt end- cut evening and symettrically
• sticky end- cuts so there is an overhang

1. put DNA sample into electrophoretic field to run gel electrophoresis
   1. positive field away from sample due to negative phosphate ions on DNA
2. smaller segments tend to migrate further away and larger segments dont migrate as far
3. place gel on salt solution buffer that has nitrocellulose filtering paper and a sponge adhering to the gel
4. DNA transfered to filter
5. hybridize with unique nucleic acid probe that is complementary to the sequence
6. remove the unbound probe
7. probe will hybridize with complementary DNA sequence
8. add ethidium bromide and view filtered paper in UV light

Its aromatic nature will allow it to fit right into the DNA (pi orbitals will allow it to do "base stacking"). This will allow the DNA to be visible in UV light.

Clinical application- how does southern blotting work for the Sickle cell anemia genome?

1. get MstII to cut the beta globulin gene
2. it will also cut at the segment at which the mutation could happen
3. if the mutation does happen, the enzyme will pass over the gene and not cut at this point
4. Results in gel electrophoresis
   1. normal Hb- one small segment and one large segment in the tract
   2. sickle Hb- one huge segment

1. take DNA fragment of interest (blunt end)
2. add a decameric linker along with ligase
3. add a restriction enzyme to cut and this will allow us to "move" the DNA sequence
4. allow us to distrupt certain genes on the plasmid and insert our desired segment

1. remover mRNA segment of interest and add an artificial primer (oligo T to go with polyA tail)
2. add reverse transcriptase and dNTPs
3. the DNA will lengthen
4. alkali digestion of mRNA template
5. attachment of oligo dG to 3' end of cDNA
6. add oligo dC primer along with DNA polymerase and dNTP's
7. you end up with a double stranded cDNA

1. add excess primers and heat to separate strands
2. cool to annel primers
3. slightly heat to synthesize new DNA
4. repeat this over and over again to amplify one desired segment

• forward (start with cDNA)
  1. insert into expression vector or deduce AA sequence
  2. transform host or prepare synthetic peptides
  3. get encoded protein or prepare specific Ab for encoded protein
• backward (start with protein)
  1. determin AA sequence, synthesize DNA probe, screen DNA library via southern blot and get cDNA
  2. prepare specific Ab, prepare DNA library, express, and screen by Western blotting to get cDNA

Process of DNA sequencing via Sanger method

1. add polymerase, primers, nucleotides, and dideoxynucleotide of interest
2. heat up to separate strands
3. cool to anneal primers
4. heat slightly to add DNA polymerase to strand
5. polymerase binds complementary nucleotides
6. while binding, polymerase will by chance ned up binding a dideoxynucleotide, so it cant continue
7. load DNA onto gel for sequencing
8. fluorescently detect each bond, so different nitrogen bases absorb at different colors

1. remove tumor tissue
2. make labeled tumor cDNA or cRNA
3. add to DNA microarray
   1. each "minislide" in the microarray reperesents a specific sequence found at a target gene (know due to human genome project)
4. comparatively analyze gene expression
5. find the molecular signature that will signify good or bad prognosis

Process of making cDNA

1. add artifical primer
2. add reverse transcripatse and dNTP's
3. alkali digestion of mRNA template
4. attach oligoG to 3' end of cDNA
5. oligo(dC) primer added with DNA polymerase and dNTP's to give us cDNA