Replication

Question 1

In all Eukaryote cells what form is DNA in?

Answer 1

Chromatin

Question 2

What is it called when DNA differs in IK?

Answer 2

tropoisomers

Question 3

State the equation that links twist, Writhe and linking number

Answer 3

Lk=Tw+Wr

Question 4

DNA has a supercoil structure
It can be right or left handed.
Which is positive and which is negative?

Answer 4

Right handed= negative

Left handed= positive

Question 5

What are the 5 forms of Histones?

Answer 5

H1 H2A H2B H3 H4

Question 6

Proteins are basic- what does this mean?

Answer 6

contains lots of positively charged amino acids which interacts with - charged DNA

Question 7

Describe the histone structure

Answer 7

2 copies of H2A H2B H3 H4 combine to form octameric, histone core around DNA is wrapped 1.6 x

Question 8

What is the role of the H1 histone

Answer 8

Linker histone binds to each nucleosome and changes conformation of DNA as it leaves the nucleosome which helps compact he DNA

Question 9

Name the model that correctly explains DNA replication?

Answer 9

Semi conservative

Question 10

Name the experiment that proved the semi conservative method

Answer 10

Meselson and Stahl

Question 11

Explain the Meselson and Stahl experiment

Answer 11

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

Question 12

Name 3 types of centrifugation

Answer 12

Sucrose Density centrifugation
Caesium Chloride Density Gradient Centrifugation
Density Gradient Centrifugation

Question 13

Describe Sucrose Density centrifugation

Answer 13

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

Question 14

Describe Caesium chloride density gradient centrifugation

Answer 14

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

Question 15

What did John Caims discover about replication?

Answer 15

Replication starts at a fixed point and is bidirectional forming the replication fork. In Euk. there are multiple forks

Question 16

Describe the replication fork and replication process

Answer 16

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

Question 17

Explain how the small fragments of DNA are joined in replication

Answer 17

Okazaki Fragments are joined by DNA ligase together in 5-3’ dir to form the lagging strand

Question 18

Name the 2 strands in DNA replication

Answer 18

Lagging Strand

Leading Strand

Question 19

What do DNA polymerases catalyse?

Answer 19

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

Question 20

What are the roles of the 2 Mg2+ during DNA synthesis?

Answer 20

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

Question 21

What are 2 important uses of DNA polymerase reactions?

Answer 21

DNA sequencing

DNA amplification

Question 22

Describe DNA sequencing

Answer 22

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 +

Question 23

What does DNA sequencing contain that has been modified and how does this affect the chain?

Answer 23

The reaction contains small amounts of modified ddNTP- Dideoxynucleotide triphosphate. Which can be added to the 3’ end but causes chain termination

Question 24

Describe Next Generation sequencing

Answer 24

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

Question 25

Describe amplifying DNA/ Polymerase Chain reaction

Answer 25

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