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Cell Replication and Manipulation (Cell Manipulation (Isolating DNA (Gene…
Cell Replication and Manipulation
Cell Replication
DNA Replication
Parental strands separate
semiconservative model
conservative model
dispersive model
Meselson and Stahl
New DNA strands grow
starts at replication fork
grows (3' OH attacks nucleotide triphosphate) --> release of pyrophosphate
Enzymes:
Preparing DNA for copying
Helicase (unwinds parental DNA strands)
Topoisomerase II: relieves unwinding
single stranding binding protein (prevents template strands from coming back together)
Copy DNA
primase
DNA poly III: synthesizes how new strand
Finishing up
DNA poly I: removes RNA nucleotides of primer from 5'end
DNA ligase
Proofreading: DNA poly I has DNA exonuclease activity
dual-function enzyme
DNA poly complexes stay in contact with each other
Lagging strand loops around
DNA replication in chromosomes
in eukaryotes (many origins of replication)
in prokaryotes (one origin of replication)
Cell Manipulation
Isolating DNA
Gene cloning
PCR
Restriction enzymes
DNA Sequencing
Sequencing by synthesis
nanopores
Editing Genes
CRISPR/Cas-9
sgRNA and Cas9 (endonuclease)
Transgenic Organisms
Plants
Crown Gall disease bacterium
T-DNA transferred into TI plasmid (tumor-inducing plasmid)
Treat plant cells with bacterium (bacterum mates with plant cells)
Plasmid: incorporated into genome permanently
Gene gun
gold particles coated with transgenes
Animals
In vitro: Somatic cell nuclear transfer (SCNT)
embryonic stem cells (derived from the embryo)