Replication Flashcards
In all Eukaryote cells what form is DNA in?
Chromatin
What is it called when DNA differs in IK?
tropoisomers
State the equation that links twist, Writhe and linking number
Lk=Tw+Wr
DNA has a supercoil structure
It can be right or left handed.
Which is positive and which is negative?
Right handed= negative
Left handed= positive
What are the 5 forms of Histones?
H1 H2A H2B H3 H4
Proteins are basic- what does this mean?
contains lots of positively charged amino acids which interacts with - charged DNA
Describe the histone structure
2 copies of H2A H2B H3 H4 combine to form octameric, histone core around DNA is wrapped 1.6 x
What is the role of the H1 histone
Linker histone binds to each nucleosome and changes conformation of DNA as it leaves the nucleosome which helps compact he DNA
Name the model that correctly explains DNA replication?
Semi conservative
Name the experiment that proved the semi conservative method
Meselson and Stahl
Explain the Meselson and Stahl experiment
Grew bacteria on heavy N15 isotope then transferred to N14 isotope.
The DNA was isolated after each generation and separated by density gradient centrifugation.
1st gen- all between N15 and N14 density
2 gen= 50% N14 50% intermediate
Name 3 types of centrifugation
Sucrose Density centrifugation
Caesium Chloride Density Gradient Centrifugation
Density Gradient Centrifugation
Describe Sucrose Density centrifugation
Samples applied to top of tube w/ gradient of Sucrose con.(More con. at bottom.)
Eventually all goes to the bottom but better separation of faction is stopped at correct time
Describe Caesium chloride density gradient centrifugation
Samples dispersed in a con. sol. of Caesium Chloride
Under high force the caesium forms a con. gradient
Samples sperate according to DENSITY
An equilibrium method
What did John Caims discover about replication?
Replication starts at a fixed point and is bidirectional forming the replication fork. In Euk. there are multiple forks
Describe the replication fork and replication process
Duplex opened up and new bases added at 3’ end
All DNA-polymerases requires single stranded DNA and all extend from a primer
Primer made from RNA and primase
Explain how the small fragments of DNA are joined in replication
Okazaki Fragments are joined by DNA ligase together in 5-3’ dir to form the lagging strand
Name the 2 strands in DNA replication
Lagging Strand
Leading Strand
What do DNA polymerases catalyse?
Catalyse the addition of Deoxynucleotide to 3’ end of new DNA molecule and requires 2 divalent mental ions (Mg2+) to bind to the active site
What are the roles of the 2 Mg2+ during DNA synthesis?
One facilitates the deprotonation of the 3’OH by a base to create a nucleophile that attacks the alpha-phosphoryl group of the incoming deoxynucleotide.
The other stabilises the - charge on the incoming deoxynucleotide
What are 2 important uses of DNA polymerase reactions?
DNA sequencing
DNA amplification
Describe DNA sequencing
DNA polymerase extends a primer which is bound to the single stranded fragment to be sequenced.
Denature and seperate products by polyacrylamide gel electrophoresis - to +
What does DNA sequencing contain that has been modified and how does this affect the chain?
The reaction contains small amounts of modified ddNTP- Dideoxynucleotide triphosphate. Which can be added to the 3’ end but causes chain termination
Describe Next Generation sequencing
Short DNA fragments= input annealed to slide using oligonucleotide adaptors,
PCR amplified to create multiple copies
Use of fluorescent nucleotides for sequencing
Image is taken after + of each new nucleotide
Use of Reversible terminator
Describe amplifying DNA/ Polymerase Chain reaction
Denature to produce single strands with heat
Add short primers that are complementary to ends of sequence
Thermostable DNA polymerase
Taq- originating from Thermus aquaticus
which can withstand the high temperature and required for DNA extension
