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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
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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)
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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
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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
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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
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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
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Southern blotting
Northern blotting
Western blotting
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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
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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
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PCR screening for cystic fibrosis
- mutant CFTR gene is shorter, but can be amplified w/ the same primer.
- will show with electrophoresis
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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
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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
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Gene therapy of adenosine deaminase deficiency
- isolate lymphocytes
- engineered retrovirus
- return the modified lymphocytes
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