Recombinant DNA Flashcards
What must a plasmid contain minimally?
- ORC
- Selection marker
- Cloning site
What enzyme cleaves DNA into fragments are to near specific recognition sites within the molecule?
Restriction endonuclease
What kind of sequence s common for a restriction site?
Palindromic
Differentiate EcoRI vs Smal enzyme cleavage.
EcoRI - “Sticky” Ends
Smal - Blunt ends
What are the steps in DNA agarose gel electrophoresis?
- Casting tray for gel slab
- Agarose solution poured into casting tray
- Comb added that forms wells for samples
- Wells loaded with samples
- Tray put in solution and attached to power supply
What is the cDNA library?
Collection of genes only encoded for proteins
What are the steps for PCR?
- Template desaturation (Heat)
- Primer annealing/binding
- Primer extension: Uses Taq Polymerase and dNTPs
Describe Electroporation, Microinjection, and endocytosis uptake of CaPO4.
Electroporation: Thousands of volts are passes across 1-2 mm of suspended cells; Increases permeability of bacteria and yeast cell membrane to drugs, chemicals, DNA, etc
Microinjection: DNA is injected into pronuclei of egg; Best time is after fertilization when there are two pronuclei; Used for gene transfer between animals
Endocytosis: CaPO4 and plasmid DNA are used to transfer DNA into host cell; Depends on cell constitution, pH, quality, and amount of nucleotide; Toxic to most primary cell lines
Describe the use of RFLP in genetic testing.
- RFLP patterns are inherited and segregate via Mendelian genetics
- size variability in detectable fragments indicate differences in the pattern of restriction sites within and around a gene
What is VNTR? What are it’s usage?
Variable number tandem repeats: Crime scene matching
Describe the inheritance and penetrance in LHON.
- mtDNA mutations (95% have one of three mutations)
- Males have much higher chance of developing symptoms than women
