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Recombinant DNA Technology
Molecular techniques that enables one to isolate, alter, and study DNA segments (genes) aka genetic engineering
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chimera
Hybrid DNA molecules that have been manipulated to contain DNA segments from two or more sources
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DNA sequencing
- The process of determining the sequence of bases along a DNA strand
- Methods: Sanger (Dideoxy) sequencing method, Radiolabeled ddNTPs
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Restriction enzymes
- Enzyme that recognizes specific base sequences in DNA and makes double stranded cuts nearby
- Three types: Type 1, Type 2, Type 3
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Type 1 restriction enzyme
Cuts at random sites distant from recognition sequence
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Type 2 restriction enzymes
- Cuts non-randomly within recognition sequence
- Recognize specific sequences and cut the DNA within the recognition sequence
- Palindromic
- Two different types of cuts: Blunt ends, staggered ends
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Type 3 restriction enzymes
Cuts at random sites near the recognition site
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Gel electrophoresis
- DNA fragments are separated on an agarose gel based on size
- Can be used to separate DNA molecules on the basis of their size and electrical charge
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Cloning vectors
- Stable replicating DNA molecules to which a foreign DNA fragment can be attached
- Three characteristics: Origin of replication (ori), selectable marker (s), and restriction site (s)
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Types of cloning vectors
- Plasmid vectors (bacteria)
- Bacteriophage (viruses)
- Cosmids (yeast)
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The Reaction Steps
- Denaturation
- Annealing
- Extension
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Induction
- The gene will be turned on or off at all times
- Induced by substrate the proteins look for
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Polycistronic message
Two types: constitutive and induction
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