DNA replication Flashcards
3 models of how DNA is copied
conservative, semi-conservative, dispersive
Meselson and Stahl experiment - what is used
E.coli, Isotopes 14N and 15N
Meselson and Stahl experiment - method
- cells transferred to medium (14N)
- DNA isolated of 1,2,3….generations
- separate DNA by density gradient centrifugation
at 0 generation
all DNA are 15N
1 generation
density between 14N + 15N (intermediate)
2 generation
14N(50%) and 50% intermediate
3 generation
14N (75%) and 25% intermediate
Density gradient centrifugation
heaviest sediment hit bottom faster
Caesium chloride density gradient centrifugation
- DNA purified from bacteria and put in caesium chloride solution
- spun in test tube for days at certain speed
- DNA form equilibrium
- higher conc of caesium at bottom
- DNA separate depending on density (buoyant density)
- sample reaches natural buoyancy in solution
- bands exposed in UV light against the film
How DNA form equilibrium
density of DNA = density of surrounding
conclusion after M and S experiment - of mixture 14N and 15N
semi conservative or dispersive because they show mix of 14N and 15N
result of redo the experiment but can be done same in denatured DNA - single stranded
semi-conservative - get 15N and 14N
while dispersive - still mixed 15N and 14N
sucrose density centrifugation
sample at top of tube containing gradient of sucrose concentration
more concentrated at the bottom
replication of E.coli
circular double stranded genome
starts at a fixed point and is bidirectional
DNA replication in Eukaryotes
has multiple replication forks which are the multiple sites
strands separate and DNA polymerase forming daughter cells
what occurs to the duplex during replication in eukaryotes
opens up and new bases added at 3’ -end
how DNA is replicated - bonding of 2 nucleotides
- deoxynucleotide triphosphate (DNT) bond to primar
- -OH in primar attacks alpha phosphate of DNT
- breaks bond and lose 2 phosphate group - hydrolysed
- 2 phosphate separate which produces energy which drives reaction of addition of individual bases
Alpha phosphate position of DNT
first phosphate close to deoxyribose
Beta phosphate position in DNT
second phosphate close to deoxyribose
Gamma phosphate position in DNT
third phosphate to deoxyribose
Lagging strand
3’ - 5’ direction of other strand template
slightly slower than 5’ - 3’
Okazaki fragments - method
- pulse labelling technique
- using radioactive labelled DNA
- pulse radioactive molecule and harvest DNA
Okazaki fragments - result
small fragments of DNA strand which can be joined together by DNA ligase
helicase
opening up DNA stand by breaking hydrogen bonds forming the multiple replication forks
primase
adding a short strip of RNA allowing polymerase to bond
polymerase II
add new nucleotides at 3’ -end in 5’ - 3’ direction
polymerase I
replace RNA laid down by primase
ligase
reform phosphodiester linkage between lagging strands to form continuous strand
binding proteins
stabilise DNA strand as it is separated as single stranded DNA is unstable
Use of DNA polymerase for DNA sequencing
extend primer bound to single stranded fragment to be sequenced
what other things needed for DNA sequencing
dideoxynucleoside triphosphate, normal dNTPs
sanger method sequencing
- add ddATP in chain which breaks the sequence at a specific point therefore have different size fragments
- denature and separate products by polyacrylamid gel electrophoresis
how to prevent formation of small fragments
have a lower concentration of ddNTP compared to dNTP
Agarose
Physical gel used as horizontal submerge
function of agarose
separate double stranded DNA fragment
how to use agarose
heated and when cooled it forms a gel substance = mesh
method of DNA sequencing using agarose gel
DNA is added and run through the gel to positive charge
shown by adding dye - fluoresces when bound to DNA
polyacrylamid gel
chemical gel
how to use polyacrylamid
gets a chemical reaction that cross polyacrylamid creating mesh network
function of polyacrylamid
for small fragments
in denaturing gel at 8M urea to separate single stranded DNA fragments
how is the DNA in polyacrylamid visualised
by autoradiography 32P or covalent attachment of fluorescent group
how to read the results of DNA sequencing
smallest fragment at the positive side reading sequences 5’ to 3’
increase in concentration of acrylamide
smaller the size of pores
ddNTPs in sequencing to be seen
different colours for different bases in different or same gel
Next generation sequencing
- fragment DNA and anneal to slide using oligonucleotide adaptors
- PCR amplified to multiple copies of that DNA
- using fluorescent nucleotide for sequencing
- images taken after adding new nucleotide
what is used next generation sequencing
reversible terminator
how reversible terminator is used
pause, take image, add nucleotide (reverse terminate)
method of reversible terminator
3’ - reversible blocking group
- after image is taken, blocking group is cleaved off and adds -OH
cluster of PCR
generate DNA
each cluster sequenced at the same time
method of cluster of DNA
nucleotide is added to DNA template - picture taken
- block and fluorescent dye removed
- repeats to next nucleotide
how DNA is amplified
using PCR - polymerase chain reaction
method of PCR
- DNA is denatured forming single strand by increasing temperature
- add short primer complementary to end of sequence of interest
- decrease temperature - anneal which binds to region to amplify
- thermostable DNA polymerase used for DNA extension
- repetition of denaturation, annealing and extension
thermostable DNA polymerase
Taq
able to withstand high temperature and used to extend DNA
Taq
originally isolated from Thermus aquaticus
how cluster of OCR is generated
computer algorithms detect signals and construct sequence
