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Lectures 23 & 24: DNA Replication & Manipulation (Replicating…
Lectures 23 & 24: DNA Replication & Manipulation
DNA is replicated in a semiconservative fashion
Helicase unwinds DNA duplex; Topoisomerase II relieves stress of unwinding
Single-stranded binding proteins stabilize single DNA strands
DNA polymerase III adds nucleotides to existing strands
RNA Primase synthesizes RNA primer at 5' end and in front of every Okazaki fragment
DNA Polymerase I removes RNA primer, puts on DNA nucleotide
DNA ligase: Joins Okazaki fragments, joins first nucleotide and rest of duplicated sequence
Lagging strand loops around to go in same direction as leading strand
DNA exonuclease helps in proofreading
Replicating chromosome ends:
Telomerase binds to 3' overhand of DNA; RNA molecule complementary to telomeric repeat, then adds telomere sequence
After, DNA polymerase adds nucleotides to other strands
Cloning a gene in bacteria:
Find gene of interest; cut plasmid and foreign DNA with same restriction enzyme- then mix and treat with DNA ligase
PCR
Denaturation (Strands split)
Annealing (& primers added)
Extension (New nucleotides added)
Reeat cycle (2^x strands)
Somatic Cell Nuclear Transfer
Nucleus of egg is removed & replaced with new one
There is a bacteria that transforms some of its DNA into plants; very ueful
Sequencing by Synthesis (SBS)
Uses Dideoxynucleotides & overlaps
Single molecule sequencing using nanopores
"Adaptor" molecule keeps bases in place long enough for charge of each nucleotide to be read
CRISPR-Cas9
sgRNA & Cas9 endonuclease cleave DNA at certain spot