Biochemistry - Unit III - Recombinant DNA

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  1. Three commonly used liquid chromatographic techniques
    • Gel filtration: based on mass; beads with pores; large proteins go around the bead and come out first; small proteins go through the bead via the pores and take longer time to come out
    • Ion-exchange filtration: based on charge; the bead is charged and the non-target protein has the same charge, comes out first, targe protein has the opposite charge and is attracted to the beads; add high concentration NaCl; target protein comes out; can reach very good separation
    • Antibody-affinity chromatography: based on affinity; very high purification
  2. SDS-PAGE
    • sodium dodecylsulfate polyacrylamide gel electrophoresis
    • separates proteins primarily on the basis of their masses
    • all proteins coated by SDS and have negative charges, move towards the positive electrode (anode)
  3. two-dimensional SDS-PAGE
    • step one: isoelectric focusing; in a pH gradient and an electrical current, proteins move to specific pH level where they are no more charged and stop moving in the electrical field; separation based on charge.
    • step two: SDS-PAGE in perpendicular direction; all proteins with the same amount of charges are separated by size in a perpendicular dimension, resulting a high degree of separation
  4. Western blotting
    • immunoblotting
    • combines several techniques to resolve and detect a specific protein
    • 1. SDS-PAGE to separate the proteins
    • 2. primary Ab is used to bind to the target proteins
    • 3. secondary Ab (more general, contains enzymatic capability) is used to bind to Ab1
    • 4. enzymatic reaction happens to locate the target protein
  5. Cleavage of DNA by the _________ EcoRI
    • restriction endonuclease
    • works with specific palindromic sequences
    • from bacteria (the bacteria also has methylase for the same sequence to protect its own genome from being degraded)
    • generates blunt/over hung (sticky) ends
    • extremely useful in genetic engineering
  6. cDNA
    • complimentary DNA
    • synthesized from mRNA (which can be isolated by chromatography using bead with polyU)
    • add polyT as primer
    • using dNTP and reverse transcriptase
    • eventually form ds cDNA
  7. Southern blotting
    Northern blotting
    Western blotting
    • DNA
    • RNA
    • protein
  8. DNA sequencing using the Sanger _____ chain termination method
    • dideoxy
    • very small amount of ddNTP mixed w/ dNTP and polymerase, primer
    • synthesis terminated whenever ddNTP is added to existing sequence
    • ordered by size in PAGE
  9. PCR
    • used to amplify a specific DNA sequence
    • 1. denature dsDNA to ss at 95°C
    • 2. anneal w/ primers at 50-60°C
    • 3. extend the primer w/ DNA polymerase at 72°C
    • 4. repeat 1-4, double the number of copies per cycle
  10. PCR screening for cystic fibrosis
    • mutant CFTR gene is shorter, but can be amplified w/ the same primer.
    • will show with electrophoresis
  11. Assay of short tandem repeat (STR) loci
    • each individual at each position has random number of repetitions of STR (2-6 nucleotides)
    • 13 CODIS (FBI) core STR loci with chromosomal positions
    • Forensic application
  12. Microarray analysis of differential gene expression using DNA microarrays (chips)
    • based on cDNA
    • determines the difference in expression of a variety site/gene
    • analyze global gene expression
    • large number of genes compared simultaneously
  13. Gene therapy of adenosine deaminase deficiency
    • isolate lymphocytes
    • engineered retrovirus
    • return the modified lymphocytes
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Biochemistry - Unit III - Recombinant DNA
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Biochemistry - Unit III - Recombinant DNA
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