DNA Replication and Manipulation Flashcards
Replication Fork
Where parental strand is splitting apart prior to synth
semiconservative model
after DNA rep, new DNA duplex consists of one old strand, one daughter strand
Conservative Model
Model suggests that after DNA rep, one duplex was 2 newly synthed daughter strand, parent strand left in tact
dispersive replication
produce 2 molecs with old and new DNA interspersed along each strand.
The Meselson-Stahl Experiment Procedure
e coli= prokaryotic replication
Grew them in medium with N15 (nitrogen isotope)
E coli incorporating N15 into their DNA (which is nitrogen rich)
Then transfer to N14 medium
SO all DNA synthed in N15 have N15 bases, DNA synthed in N14 have N14 bases
Parents N15, daughters N14
N14 lighter than n15
Centrifuge: b/c N15 heavier.
N15 go farther in test tube because heavier than N14
The Meselson-Stahl Experiment Results
Result after 1st Generation: EITHER semiconservative rep or Dispersive rep. Because they were at hybrid densities
Result after 2nd Gen: Has to be semiconservative replication.
b/c more strands that are just N14 (b/c they are new “parental” strands)
How was it figured out that eukaryotes also semiconservative model?
Experiments with fluorescently labeled DNA led to the discovery that eukaryotic cells also replicate semiconservatively
half labled with flourescents
Template Strand
- READ 3’ to 5’ so daughter can be synthed 5’ to 3’
- Break off 2 phosphate fround
- 3’ OH of growing strand attacks high nrg phosphate bond of incoming nucleotide, providing nrg to drive rxn
Discontinous Replication
Lagging Strand
Replication opposite direction of unwinding
Multiple RNA primers
RNA Primers
5’ end of each daughter cell
Allows for DNA Polymerase to come in and synthesize
DNA requires a short strand of double stranded nucleic acid for it to synthesize anything
Laid down by RNA primase
many in lagging strand = okazai fragments
Okazaki Fragments
Primer removed and replaced with DNA, and fragments of discontinuous (lagging strand) are ligated (sewn together) where they meet
proofreading
DNA Polymerase
bulge b/c bases are different sizes, and it catches this
Cleavage
When a little nucleotide is cut out and replaced with the right one
Replisome
where everything happens (area of replication fork)
Helicase
Topoisomerase II
Single Stranded Binding Proteins
Helicase
Unwinds DNA duplex and sends it in different dirctions
Build up tension further down molec
Topoisomerase II
relieves stress further down strand of unwinding so helicase can come by and unwind it
Single Stranded Binding Proteins
stabilized single strands of DNA
Protects from attack
Makes sure DNA parent strands don’t sew back up
Origin of Replication
Multiple ones
Where replication fork is
forks moving in opposite directions
replication bubbles
DNA polymerase complex
site of growing chain length in one DNA subunit at a time, checking for errors as it goes along
Leading strand
the new strand with the free 3’ end
Lagging Strand
one with the free 5’ end
Circular DNA
Bacteria, mitochondria, chloroplasts
Replication of Circular DNA
ONE ORIGIN OF REPLICATION
2 rep forks going in either direction
okazaki fragments, leading and lagging strand
End Replication Problem
o Lagging strand, constantly laying down new RNA primers
• No place to do it when you reach end of DNA
• Cant generate new DNA, cant add new primer
• Last RNA primer laid down 100 base pairs away from the end
• End up with un replicated bit of DNA at the end,
• A daughter strand doesn’t make it all the way to the end of the parent strand from which it was synthesized.
• So will always lose some base pairs
o New daughter strand will be shorted about 100 nucleotides in every round of replication.
o One day they wont be able to divide again, hang out in G0 forever
Adult somatic cells: mitotic division only 50 times
reason for aging?
Telomerase
Solution to end replication problem
GERM CELLS ONLY (healthy)
in somatic cells, teomerase turned on leads to cancer
Germ Cells
- Telomerase enzyme contains an RNA template that allows the template strand to be lengthened by telomere repeats.
- Germ line cells are “immortal”
telomeres
long stretch of repetitive DNA at end of somatic cells to deal with end replication problem
so you lose repeat, not important genes, with division
Polymerase Chain Reaction: PCR general info
polymerase from themoatically stable organism (one that lives in hot environments)
targeted region of DNA to be replicated/amplified into as many copues as desired
Gel electrophinesis
Swimming Pool ex (SI)
• DNA samples are inserted into wells at one edge of gel
• Pass electricity through (also DNA matrix and liquid buffer)
• DNA migrates to positive charge at opposite end of chamber
• Passes through substrate depending on size
• Small pieces pass rapidly
• Numbers on pictures are wells
Bigger stay at negative, little go to positive
Southern Blot
restriction enzymes used to cut up whole DNA
separate using Gel electrophinesis
pass DNA from gel to filter paper
look for where fragment of interest migrated
probe filter paper with radiolabeled matching gene probe
expose film, see where bands migrated
can use flourencent nucleotides instead of raido
Polymerase Chain RXN: process for PCR
Denaturation
Annealing
Extension
PCR Denaturation
Heat up strands and they come apart
Unwinding
PCR Annealing
When cooled, two primers provided to anneal to their complementary sequences on the DNA template strand
o need temp not too low, or DNA will zip back together
PCR Extension
Synth new DNA strands
Extend primers in a 5’ to 3’ direction
PCR Components
o Template DNA
o DNA Polymerase (TAQ – polymerase from Thermus aquaticus)
o The four deozynucleoside triphosphates (A, T, G, or C)
o Two primers (short stretches of DNA).
More PCR
o Target specific segement of DNA
• 1. Template duplex often longer than amplified region
o provide primers that match on either side of DNA
o let amplification occur
o end up with 2 copies: parental and new
o Go through again, end up with 4 copies
o After n cycles of amplification there are 2^n copies of the template sequences.
o Shorter copies in each subsequent round
Restriction Enzymes
Cut DNA at specific sequences
allows:
• pieces from the same or different organisms to be brought together in recombinant DNA technology
• or can cut large genomes into small pieces for further analysis
Blunt ended cut
Even on either side of cut
Overhang cut
Overhang on either side of cut
Sanger Sequencing: Terminator Nucleotides
Don’t have 3’ hydorxyl group, just hydrogen, so others cant add on
Sanger Sequencing- The Sequence
o Run fragments out on gel
o Read gels looking at readout
o Florecnese peak tells us what last nucleotide was
o Read one fragment at a time, put all together, find sequence of daughter strand
Recombinant DNA
usually e. coli used Transformed Take up DNA from surrounding environment "Heat shock" makes them stressed and suck up plasmid. Has its genome and the one you gave it.
Donor DNA
The one we add in
PCR replicate it (double stranded from us sometimes)
Ligate to vector with ligase of sticky ends
Vector DNA
circular pieces of DNA taken up. From e. coli.
Cut it
Ligate with sticky ends to Donor
GMO examples
Gene gun
engineer viruses to insert into eukaryotic genomes
maybe review this
Why use Sanger?
Don’t know template, so make daughter to ID parent
Why Recombinant DNA?
to replicate the gene you want.
What to do with recombinant dna?
make new type of e. coli cells with its genome and recombiant plasmid
Make lots of it to replicate the gene of interest
Southern Blot
Pass DNA from gel electro to filter paper
want to know where DNA fragment of interest is
Probe filter paper (probe matches gene)
visualize by exposing film
