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BIO1334 Genetics > DNA Replication, PCR & Cloning > Flashcards

DNA Replication, PCR & Cloning Flashcards

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Biology 101 flashcards
1
Q

What is the S phase of the cell cycle?

A

DNA replication.

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2
Q

Give the 4 stages of the cell cycle.

A

G1, S, G2, M.

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3
Q

What are telomeres?

A

Highly repetitive sections of DNA that protect the ends of chromosomes from degradation, recombination & fusion with other chromosomes.

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4
Q

What is the centromere?

A

Repetitive DNA which forms the spindle attachment site in mitosis.

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5
Q

What is the origin of replication?

A

Site where duplication of DNA begins, each chromosome will have many origins.

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6
Q

What is semi-conservative replication?

A

The new DNA helix has one new strand and one old strand.

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7
Q

What is needed for DNA synthesis?

A

DNA polymerase & Mg^2+
dNTPs
Single stranded DNA template.
Primer 3’ -OH

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8
Q

Describe the process of DNA replication.

A

DNA polymerase unwinds the double helix.
Single stranded binding protein prevents base-pairing until DNA polymerase arrives, they leave one face open so it is still accessible to DNA pol.
Primase synthesises short RNA primer copied from DNA.
Sliding clamp loaded onto DNA by clamp loader enzyme, opens up the sliding clamp allowing it to encircle the DNA, and it is then locked around the DNA by ATP hydrolysis, this also releases clamp loader.
DNA polymerase elongate RNA primers with new DNA.
Nucleases remove RNA at 5’ end of neighboring fragment & DNA polymerase fills the gap.
DNA ligase connects adjacent Okazaki fragments on lagging strand.

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9
Q

What is the directionality of DNA synthesis?

A

New strand synthesised 5’ to 3’

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10
Q

Why are replication bubbles necessary in eukaryotes?

A

Multiple points of origin are needed for each chromosome as replicating the whole chromosome from one origin would take too long.
Bubbles meet eventually & replication is complete.

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11
Q

Describe the process of bacterial replication.

A

One origin.
An intermediate theta structure forms, the replication forks move away from the origin & eventually meet.
Both strands are copied at fork.
Synthesis of new strand 5’ to 3’

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12
Q

What is the directionality of the leading strand?

A

5’ to 3’

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13
Q

What is the role of histone chaperones in eukaryotic DNA replication?

A

Load histones onto newly-synthesised DNA.

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14
Q

What is the problem caused when helicase unwinds the DNA helix and how is it fixed?

A

Causes supercoiling ahead of the replication fork.

Topoisomerases unwind this supercoiling by breaking & reforming phosphodiester bonds using a swivel motion.

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15
Q

Give the rate of DNA replication in eukaryotes vs E.coli.

A

~50bp s^-1 : ~1000bp s^-1

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16
Q

What is the combined accuracy of eukaryotic DNA replication?

A

1 in 10^10

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17
Q

Why is DNA pol. more accurate than RNA pol.?

A

DNA pol. must undergo a conformational change to close around the DNA strand, so if base pairing is wrong the conformational change is less likely to occur.
DNA pol. can also proofread when it ‘chews back’ to remove an incorrect base pair an replace with the correct one.

18
Q

What is the function of mismatch repair proteins?

A

MutS cans newly synthesised strands & detects kinks caused by mismatched base pairs.
MutL binds to a nick in newly synthesised strand, The DNA between the nick and the kink is removed & then replaced by correct sequence.

19
Q

What are errors in mismatch repair proteins are associated with?

A

Predisposition to some cancers.

20
Q

What is PCR?

A

Polymerase chain reaction, amplification of DNA.

21
Q

What does PCR require?

A
  • template DNA
  • primers
  • dNTPs
  • Mg 2+ buffer
  • Taq polymerase (from organisms that live in very hot environments)
22
Q

Give the 3 steps in PCR.

A

Denaturation, double DNA strand melts open at 95 degrees C.
Annealing, primers bind to DNA and polymerase attaches and starts copying DNA at ~50 degrees C.
Extension, at 72 degrees C Taq polymerase can extend the fragment from the primers.

23
Q

How many cycles does PCR go through?

A

~30-40

24
Q

How can you calculate the number of DNA copies for a certain PCR cycle number?

A

2n (n = cycle no.)

25
Q

Give the limitations of PCR.

A

Only the DNA between primers is amplified
sequence information is required to design 2 primers
Limit on length of amplified fragment
Potentially high error rate
Very sensitive to exact reaction conditions, not easily quantified
Tiny amounts of contaminating DNA will also be amplified

26
Q

How are PCR primers made and how long are they?

A

Commercially, around 20bp long.

27
Q

What feature does gel electrophoresis separate DNA by?

A

Size.

28
Q

Which electrode does DNA flow towards in gel electrophoresis?

A

+ electrode (cathode)

29
Q

What effects how far/fast DNA moves toward the cathode in gel electrophoresis?

A

Conformation - linear/circular/supercoiled

Size

30
Q

How is the DNA in gel electrophoresis seen?

A

Dyed with fluorescent dye for detection by UV exposure.

31
Q

Give the uses of PCR.

A 
DNA sequencing
Detection of pathogens in water, clinical samples etc
Genetic fingerprinting
Forensic analysis
Forensic analysis
Diagnosis of genetic disorders
Prenatal diagnosis
Analysis of ancient DNA
32
Q

How can the products of PCR be directly sequenced?

A

Pyrosequencing.

33
Q

What are plasmids?

A

Extrachromosomal circles of DNA in bacteria that can replicate and be exchanged between lineages?

34
Q

How are plasmids used in recombinant DNA technology?

A

Plasmid extracted from bacteria.
Restriction endonucleases recognize specific cutting site & cuts leaving a 4 base overhang.
DNA fragment to be cloned is cut with same restriction enzyme.
DNA ligase sticks them together.

35
Q

Give the function & requirements of DNA ligase.

A

Forms phosphodiester bonds
Requires ATP
Allows 2 DNA molecules to be joined (recombinant DNA)
Allows genes to be inserted (stuck into) plasmids

36
Q

Give the overall process of gene cloning?

A

Introduce recombinant plasmid into bacterial cell
Cell will divide
Clone of cells
Recover DNA for analysis
Can also be done by insertion into bacteriophage DNA vectors

37
Q

Give the purpose of gene libraries.

A

Collection of recombinant clones

Can screen for clones containing gene of interest

38
Q

What is transgenics and how is it possible?

A

Take a gene from one organism & express it in another organism.
Genetic code is universal.

39
Q

How can DNA be expressed in foreign cells?

A

Introduction of DNA into cell
Replication & expression in the cell
For expression of eukaryotic DNA in a prokaryote you need to make a cDNA copy of mRNA (introns).

40
Q

Give the useful products of genetic engineering.

A 
Human insulin
Blood clotting factor VIII
Human growth hormone
Bovine chymosin
Hepatitis B vaccine
Artemisinin (anti-malarial)
41
Q

What are reporter genes and their function?

A

Proteins tagged with GFP

Illustrates particular gene fused with GFP, can now see where gene is localised

42
Q

Give an agricultural application of synthetic biology?

A

Artemisinin (antimalarial) production in yeast faster than through crop production & limiting factors of crop production.

BIO1334 Genetics (15 decks)

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