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asdfghjmak 2 > P1 L2 > Flashcards

P1 L2 Flashcards

1
Q

What was Waston and Crick’s theory

A

The semi-conservative replication:
One strand serves as a template

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2
Q

What are the three models of DNA replication proposed by Meselson and Stahl?

A

Semiconservative, conservative, and dispersive replication

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3
Q

What happens in the conservative replication model?

A

The entire original DNA double helix serves as a template for synthesis of a new double helix (copy of old + new)

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4
Q

What happens in the dispersive replication model?

A

The original DNA double helix breaks into fragments, each serving as a template for new DNA fragments

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5
Q

In semiconservative replication, what serves as a template?

A

Each STRAND of DNA serves as a template for synthesis of the complementary strand

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6
Q

What experiment did Meselson and Stahl perform in 1958?

A

They grew bacteria in heavy nitrogen and transferred them to light nitrogen to differentiate the models of DNA replication

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7
Q

What did the presence of heavy DNA indicate after one round of replication?


A

That the DNA band is a bit lighter, indicating semiconservative replication

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8
Q

What is the outcome after two rounds of replication in semiconservative replication?

A

Another band appears that is even lighter

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9
Q

Where does the replication of bacterial chromosomes begin?

A

At the origin of replication, labeled as oriC

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10
Q

Explain the replication of the bacterial chromosome.

A

It starts at OriC. 2 enzymes move in opposite directions and both DNA strands are replicated at the same time. Since the strands are antiparallel, a replication fork is needed.

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11
Q

How does DNA replication proceed after it begins at oriC?

A

It proceeds bidirectionally with two replication forks

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12
Q

In which direction does the DNA polymerase synthesise

What is the function of DNA polymerases during replication?

A

Synthesis from 5‘ –> 3’.

DNA polymerases add bases only at the 3’ end of the DNA

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13
Q

What initiates DNA synthesis?

A

An RNA primer synthesized by primase
Existing hydroxyl groups at the 3’ end are required so that nucleotides can be added - provided by primer

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14
Q

How does semi-discontinuous replication work?

A

The bases are always attached at the 3’ end.
In the lagging strand, this leads to synthesis proceeding in the opposite direction -> away from the replication fork and in fragments

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15
Q

What is the difference between the leading strand and the lagging strand during DNA replication?

A

The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously

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16
Q

How is the replication fork structured?

A

Tomoisomerase and helicase

on the leading strand:
RNA primer
DNA polymerase III
Single-strand binding proteins

On the lagging strand:
Okazaki fragment
multiple RNA primers
Primase opposite of CTG
DNA polymerase III

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17
Q

Roles of Tomoisomerase

A

Tomoisomerase - removes supercoils

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18
Q

Role of DNA helicase

A

Helicase - separates strands of DNA
It separates the strands by forming a ring around a strand and pushing it through the ring. In the process, ATP becomes ADP.

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19
Q

Role of SSBs (single-stranded DNA binding proteins)

A

They bind and stabilise single-stranded DNA

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20
Q

What is the function of the clamp loader in DNA replication?

A

The clamp loader holds the proteins across the replication fork and loads the clamp protein

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21
Q

How long is the primase and how often does it occur?

A

It occurs only once on the leading strand and every 1000-2000 nucleotides on the lagging strand. It is 10 nucleotides long.

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22
Q

What are Okazaki fragments?

A

Short fragments of DNA synthesized on the lagging strand

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23
Q

How are the Okazaki fragments put together? (3 steps)

A
  1. the DNA polymerase III continues to the next RNA primer.
  2. polymerase I degrades the RNA primer and synthesises the missing piece of RNA
  3. the DNA ligase then links the hydroxyl group (3‘) with the phosphate group (5’) of the other piece of DNA.
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24
Q

Role of DNA Ligase?

A

DNA ligase links the three prime hydroxyl group and the five prime phosphate of two DNA fragments

25
Q

What is the overall process of DNA replication in bacteria?

A

Initiation at oriC
Bidirectional replication
Continuous synthesis on leading strand
Discontinuous synthesis on lagging strand
Termination at ter sites

26
Q

What is the error rate of DNA synthesis?

A

1 / 100,000 base pairs

27
Q

Can all DNA polymerases ‘proofread’?

A

No

28
Q

How does proofreading work?

A

If an incorrect base pair is formed, a base pair is added first:
1. the incorrect base is removed by the 3‘ to 5’ prime exonuclease activity.
2. the correct base is inserted.

DNA polymerases can reverse by one base pair to excise incorrect bases

29
Q

How much does proofreading increase the accuracy of DNA replication?

A

It increases accuracy by about 100-fold

30
Q

What are the main steps in the initiation of DNA replication at oriC?

A

DnaA binds to oriC
Local melting of AT-rich DNA occurs
Helicase unwinds the double helix
SSB stabilizes single-stranded DNA
Primase synthesizes short RNA primer

31
Q

What is the ‘generation time’?

A

The time a bacterium needs to divide:
25-70 min
depends on medium

32
Q

What is the replication time for E. coli?

A

40 minutes constantly

33
Q

Describe the slow growth rate of bacterial DNA replication.

A

I (Initiation time): 70 min
C (replication time) : 40 min
D (Division time): 20 min
It is a cycle, the initiation time consists of the C and D of the previous replication (40+20)<70

enough time to finish processes

34
Q

Describe the ‘fast growth rate’ of bacterial DNA replication.

A

I: 30 min
C: 40 min
D: 20 min

The replications overlap here, as replication begins before initiation is complete.

chromosome is already replicating when daughter cells receive them.

35
Q

How is chromosome replication completed? (3 steps)

A

The terminus is located opposite the ‘origin’ and has 2 sides: terA and terB.

1) The Tus protein (terminus utilisation substance) blocks DNA helicase activity.

2) This allows the replication fork to go in one direction only.

3) This causes the replication forks to meet somewhere between terA and terB and the process is stopped.

36
Q

Arthur Kronberg

A

He isolated DNA polymerase I from E. coli and showed that this enzyme can synthesise DNA in vitro.

37
Q

F. Sanger

A

DNA sequencing technique

38
Q

K. Mullis

A

PCR technique

39
Q

What are the required parts of Sanger sequencing? (5 points)

A

-single-stranded DNA template

-DNA polymerase

-DNA primer

-deoxynucleoside triphosphates (dNTPs)

-modified di-deoxynucleoside triphosphates (ddNTPs)
-> sequencing of the complementary strand

40
Q

What is the significance of modified bases ddNTPs in Sanger sequencing?

A

They stop the reaction by lacking the 3’ hydroxyl group required for extension

41
Q

What happens when a modified variant of the normal dTTP is added during in vitro synthesis of DNA? (Sanger sequencing)

A

The addition of ddTTP leads to a cancellation of an A. However, the cancellation always occurs at a different A -> different lengths

42
Q

How are chain terminations in Sanger sequencing characterized?

A

Chain terminations are random, resulting in fragments of increasing length.

-> sequence entire human genome

43
Q

How do the ddNTPs improve data utilisation?

A

The four ddNTPs are labelled with fluorescent colours. The fragments themselves are separated by gel electrophoresis. The colours make it possible to do all 4 at the same time.

44
Q

What does PCR stand for? What is the aim?

A

Polymerase chain reaction
The multiplication/amplification of specific DNA fragments.

45
Q

Describe the PCR process (3 steps)

A
  1. denaturation: the strands are melted (94 °C)
  2. primer binding: the primers are attached to the DNA strands (50°C)
  3. synthesis: The DNA polymerase extends the 3’ ends -> synthesis of the counterstrand (70°C)

4.Repeat the cycle for amplification.

46
Q

Which sequence denatures more easily?

A

A and T

47
Q

How quickly does PCR replication occur?

A

doubled the first time -> then exponential increase.

30 cycles -> 2^30 = 1.1 billion

48
Q

Why is the DNA polymerase not destroyed during denaturation?

A

94+C, the enzymes of termophilic bacteria or archaebacteria survive this without problems

49
Q

Taq Polymerase

A

: A heat-stable DNA polymerase isolated from the bacterium Thermus aquaticus, commonly used in PCR.

50
Q

What is the condition for PCR to work?

A

The short DNA sequences must be known

51
Q

What is the principle of top-down sequencing?

A

The genome is divided into smaller and smaller parts -> with Sanger sequencing

52
Q

What is the principle of shotgun sequencing?

A

The whole genome is randomly divided into different fragments. Sanger sequencing results in overlapping sequences and the entire genome can be analysed.

53
Q

What is comparative genomics?

A

Comparison of genomic features among different organisms.
Identifying similarities and differences.
Understanding evolutionary relationships.

54
Q

What is the next generation sequencing approach?

A

Sequencing-by-synthesis approach.
Massive parallelization of sequencing reactions.
Sequencing billions of sequences at once.

55
Q

What is the role of bridge amplification in next generation sequencing?

A

It synthesizes new DNA strands that cluster together on a glass slide.

56
Q

How does nanopore sequencing work?

A

Single-stranded DNA passes through a pore and blocks the flow of ions. Current monitored. Diff size base = diff current

57
Q

How does single-molecule real-time sequencing work?

A

It monitors incorporation of fluorescently labeled bases during DNA synthesis.

58
Q

What are the advantages and disadvantages of nanopore sequencing?

A

+ Inexpensive and small device
+ can read for a long time
- High error rate
based on base modification

59
Q

What are the advantages and disadvantages of single-molecule real-time sequencing?

A

+ can read for a long time
+ slightly better quality
- very expensive
based on base modification

asdfghjmak 2 (8 decks)

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