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BIOL1007 > DNA > Flashcards

DNA Flashcards

1
Q

What is a gel?

A

A gel is a water-retaining polymer network

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2
Q

Give 5 examples of some biological gels

A
  1. Cartilage
  2. vitreous humour in eyeball
  3. Cytoplasm
  4. Hydrogel in nuclear pores
  5. Polysaccharides in seaweed
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3
Q

What does the ‘central dogma’ explain?

A

The flow of genetic information

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4
Q

Give me some examples of what proteins are involved in?

A
  1. metabolism
  2. structure
  3. function
  4. organisation
  5. regulation
  6. development
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5
Q

What did Friedrich Miescher discover in 1869? and what did he name it?

A

He found acid molecules which were rich in phosphate and isolated from cells in the puss.

He called it ‘nuclein’ (DNA)

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6
Q

Whats the location of DNA in Eukaryotes and Prokaryotes?

A

Eukaryotes:

in the nucleus in chromosomes (complexes of proteins and DNA –> chromatin)

Prokaryotes:

Not membrane bound, circular, supercoiled-naked DNA (not bound in a protein coat)

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7
Q

State some comparisons between prokaryotes and eukaryotes?

A

Prokaryotes

  • little internal structure
  • 2 um in diameter
  • 70s ribosomes

Eukaryotes

  • membrane bound organelles
  • 20 um in diameter
  • 80s ribosomes
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8
Q

Does the genome size vary between species?

A

Yes

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9
Q

Roughly how many base pairs does the human genome have?

A

3 billion base pairs in the human genome

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10
Q

Do more chromosomes means a more complex organism?

A

No

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11
Q

what’s the relationship between the number of chromosomes and the number of genes?

A

there is no relationship

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12
Q

Label this chromosome

A
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13
Q

What is the product of cell division in humans?

A

2 diploid cells, each with 23 pairs of chromosomes

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14
Q

What is meant by the term ‘junk DNA’?

A

genomes of eukaryotes which contain DNA of unknown functions

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15
Q

How many copies of each chromosomes does a human cell contains?

A

2 copies

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16
Q

Whats means by the term Homologous chromosome?

A

1 paternal and 1 maternal chromosome, that contains the same gene at the same loci

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17
Q

Whats the Karyotype?

A

A full set of chromosomes

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18
Q

Whats an abnormal karyotype? What can these lead to?

A

Any chromosomes number that deviates from the normal set of chromosomes aka. Karyotypes.

This can lead to genetic disorders

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19
Q

Name 6 genetic disorders

A
  1. Down syndrome
  2. Turner syndrome
  3. Klinefelter syndrome
  4. Leukaemia
  5. Cystic fibrosis
  6. Sickle-cell disease
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20
Q

What is the reason that one may have down syndrome?

A

their cells have 3 copies of chromosomes 21 (Trisomy 21)

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21
Q

In 90% of down syndrome cases, what parent does the extra chromosome tend to come from?

A

The mother

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22
Q

Whats turner syndrome?

A

Women with turner syndrome only have 1 X chromosome (this is an example of monosomy: where only 1 parent contributes the X chromosome)

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23
Q

In 75-80% of turner syndrome cases, what parent does the extra X chromosome come from?

A

the mother

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24
Q

Whats Klinefelter syndrome?

A

Men with Klinefelter syndrome have 1Y and 2X chromosomes (trisomy)

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25
Q

With Klinefelter syndrome, what parent does the extra X chromosomes come from?

A

it can come from either parent

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26
Q

What are the genetic conditions of downs, turner and klinefelter mostly caused by? what is this?

A

non-disjunction: where a pair of chromosomes fails to seperate during formation of gametes

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27
Q

What chromosomes is Leukaemia linked to?

A

the Philadelphia chromosome

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28
Q

In Leukaemia, what leads to the formation of the BCR-ABL oncogenic fusion gene?

A

The translocation of material between chromosomes 9 and 22

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29
Q

What chromosome is the CFTR gene found on?

A

chromosome 7

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30
Q

Whats the most common mutation that causes CF? what happens in this mutation?

A

∆F508

this is the deletion of 3 nucleotides that reuslts in the loss of an amino acid

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31
Q

What’s the role of a normal CFTR channel? What happens in a mutant CFTR channel?

A

Normal CFTR channel:

Moves Cl- to the outside of the cell

Mutant CFTR channel:

Doesn’t move Cl- outside of the cell. Leads to sticky mucus build up on the outside of the cell

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32
Q

Whats does sickle-cell disease affect?

A

Haemoglobin

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33
Q

Whats different with the strucutre of the cells containing haemoblobin with the people who have sickle-cell disease?

A

They have atypical haemoglobin molecules

The RBC are cresent shaped

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34
Q

Tell me the strucutre of haemoglobin

A
  • 2 alpha and 2 beta subunits
  • contains a haem group with a Fe2+ group
  • quaternary structure
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35
Q

Name the genes that code for alpha and beta globin proteins

A

HBB codes for beta-globin

HBA codes for alpha-globin

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36
Q

Tell me about the main mutation in base codes that leads to sickle cell disease

A

A is chnaged to T at position 17 in the beta chain

This changes the codon GAG (glutamic acid) to GTG (valine)

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37
Q

Tell me some symptoms of sickle-cell diease

A
  • anaemia (low number of RBC)
  • frequent infection
  • episodes of pain
  • painful swelling of hands and feet
  • vision problems
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38
Q

In 1928, what did Griffith set out to find out?

A

Whether DNA was the genetic material in cells

39
Q

What did Griffith undertake his experiments with and what did he discover?

A

Undertook his experiments with Sterptococcus Pneumonia and injected it into mice

Discovered that the non-virulent strains become virulent when mixed with heat-killed virulent strains

this transformation was permanent which proved that something from dead cells was being transformed in living cells

40
Q

Whats does the following mean?

  1. S strain (smooth strain)
  2. R strain (rough strain)
A

1. S strain: has a polysaccharide coat which helps invade the immune system and its hard to fight infection

2. R strain: has no polysaccharide coat which makes it prone to being cleared by immune system. this enables mice to get rid of bacteria

41
Q

Tell me the steps to Giffith’s experiment and what principle he determined?

In 1944, what scientist then decided to discover the transforming principle?

A
  1. Killed the S-strain with heat and mixed with R-strain
  2. This made an S strain
  3. This is because something transformed R–> S strain (this proved the transformation principle)
  4. All descendants of the transformed bacteria were also infectious
  5. Griffith didn’t know what component of the heat killed S-strain was responsible for the change. He called this the transforming principle

In 1944, Avery set out to find the transforming principle

42
Q

Tell me the steps to Avery’s experiment and how they detemined that the transforming principle was DNA

NOTE: there were LOTS of steps :(

A
  1. Broke open heat killed bacteria with a detergent which released contents of cell
  2. Used a centrifige to seperate cellular components
  3. To ensure the extract contained the transforming principle, the extract was mixed with a living R bacterium
  4. Mixture was incubated and plated in a petri dish
  5. Colonies of S bacteria appeared on dish
  6. This indicated that the R bacteria has been transformed

​This allows Avery to figure out that a component of the extract was responsible for the transformation

Extract components: Polysaccharides, Proteins, DNA, RNA

  1. They developed a method to isolate each component and test each one for its ability to transform the bacteria
  2. First, the extract was mixed with an enzyme called S3 (this removed the polysaccharides)
  3. sample treated with live R bacteriumand incubated on petri dish
  4. S colonies grew. Therefore transformaiton had occurs without polysaccharides, meaning that was not the transforming principle
  5. Next, the extract was treated with protease (removed the proteins)
  6. Sample mixed with live R bacterium and incubated in a petri dish
  7. S colonies grew, so proteins were not the transforming priniciple
  8. The remaining extract contains no Polysaccharides or proteins and only contained DNA and RNA
  9. RNase was used to break down the RNA in the extract
  10. A sample of extract containing only DNA, was mixed with live R bacterium and incubated in a petri dish
  11. S colonies grew meaning that RNA was not the transforming principle
  12. Nucleic acid mixture was treated with DNase which removed DNA
  13. Sample containing only RNA was mixed with live R bacterium and was incubated in a petri dish
  14. No S colonies grew meaning that DNA must have been the transforming principle
43
Q

In 1952, what did Hershey and chase set out to find?

A

Whether it was the protiens of nucleic acids that were responsible for passing along genetic material

44
Q

What did Hersehy and chase use for their experiments and why?

A

they used bacteriophage’s as they have many advantages

45
Q

Tell me the advantages of using Bacteriophages?

A
  1. they reproduce quickly
  2. made up of only nucleic acids and proteins (meaning they dont contain any lipids or carbohydrates)
  3. have a protein coat on the outside and nucleic acids on the inside
46
Q

Tell me the stages to the reproduction of bacteriophages?

A
  1. attach to outside of host cell
  2. inject their genetic material into the cell
  3. protein coat of bacteriophage stays on outside of host cell whilst the genetic material goes into the host cell
  4. Bacteriophages genetic information halts the host cells functions
  5. host cell starts to produce copies of bacteriophage
  6. offspring and parent are gentically identical
  7. host cell become porous and releases all of the new bacteriophages out which allows them to infect even more cells via the same method
47
Q

Hershey and chase split the bacteriophages into 2 groups. One testing the protein coats and one testing the nucleic acids.

What did they label (radioactively label) the protein coats and nucleic acids?

A

Protein coat was labelled with

35S

Nucleic acid was labelled with

32P

48
Q

Tell me the steps to Hershey and Chases experiment

A
  1. allowed the 2 groups of bacteriophage (T2) to infect the host cell (E.Coli)

This way they would know what component (protiens or nucleic acids) were responsible for inheritance as it would produce radioactive offspring

  1. The bacteriophages injected their genetic material into the host cell

the genetic material from the 35S would not produce radio acitve offspring as genetic material no labelles

the genetic material from 32P would produce radio active offspring

  1. They ‘blended’ the host cell to remove the protein coats from the outside of the host cells
  2. The protein coats dissolved in the liquid portion of the experiment, which is called the Supernatant
  3. the solution is centrifuged to seperate the mixture into a liquid portion, containing the protein coats, and a solid portion containing everything else. the Solid portion is called the pellet
  4. this experiment showed that nucleic acids are responsible for inheritance
49
Q

Tell me the strucure of DNA

A

DNA contains

  • pentose sugar- deoxyribose
  • phosphate group
  • organic nitrogenous bases: arginine, cytosine, thymine and gyanine
50
Q

What bases are classed as the pyrimindines and the purines? How many rings do they have in their strucure?

A

Adenine and gunaine are purines, they have a double ring strucutre

Thymine and Cytosine are pyrimidines, they have a single ring structure

51
Q

Identify the nitrogenous bases…

A
52
Q

On DNA, the bases are located on the inside of the helix and the phosphates are on the outside, what does this mean for the polarity of DNA?

A

The inside is hydrophobic and the outside is hydrophiic

53
Q

What are the 2 major bonds in DNA and what are they between?

A
  1. Phosphodiester bond: this is between the phosphate group of one nucleotide and the sugar residue of the next nucleiotide

2. Beta N-linked glycosidic bond: a beta-glycosidic bond is between two carbons with different sterochemistry and a glycosidic bond is between a carbohydrate and another molecule

54
Q

What was Chargaff’s rule?

A

His rule stated that DNA from ant call of any organism should have a 1:1 ratio of pyrimidine and pyrine bases and that A=T and C=G

55
Q

Why don’t bacteriophage follow Chargraff’s rule?

A

They have a single stranded DNA

56
Q

What did Roslaind Frankline and Maurice Wilkins do and what did they discover about the strucure of DNA?

A

What they did:

  • dehydrated DNA and surrounded it with water to look how the strucutre chnages with different levels of water availability
  • obtained X-ray diffraction patterns on DNA fibres

What they discovered:

  • 10 repeting units per arm
  • X shape produced shows a helix strucure
  • gap at position number 4 suggest’s that theres a slight offset and theres interference which shows the presence of the major and minor groove of DNA
  • 0.34 nm between nucleotides (3.4 Å)

NOTE: 1Å (angstrom)= 10-10 m

57
Q

What did James Watson and Francis Crick discover?

A
  • Bases were on the inside
  • phosphates on outside
  • between A and T there’s 2 hydrogen bonds
  • between G and C there’s 3 hydrogen bonds
  • bases are hydrophobic
  • base pairing is complementary
  • two strands are anti-parellel
  • right handed double helix
  • base pairs are perpendicular to the helix axis
  • centre of helix runs through base pairs
  • known as B-DNA
    *
58
Q

Tell me the key features of B-DNA?

A
  1. right handed
  2. 0.34 nm between base pairs
  3. 3.4 nm per turn
  4. 10 bp per turn
  5. roughly 2 nm in distance
  6. has 2 grooves of unequal size
59
Q

Tell me all the possible combinations of DNA possible?

A

A-DNA

Z-DNA

B-DNA

60
Q

Tell me the features of A-DNA?

A
  • right handed
  • formed when DNA is dehydrates
  • wider
  • bases tilter
  • offset form axis
61
Q

Tell me the features of Z-DNA?

A
  • left handed
  • formed by some GC-containin sequwnces at high salt concentrations
62
Q

Identify which DNA conformation these are…

A
63
Q

Whats the hyperchomic effect of DNA ?

A

This effect is the increase in absorbance of a material for instance DNA with UV light which leads to the denaturing of the DNA

64
Q

What has a higher absorbance, single or double stranded DNA and why ?

A

single stranded has a higher absorbance as the bases and no longer stacked on each other

65
Q

Whats a decrease in absorbance called?

A

Hypochromicity

66
Q

do high salt concentrations give an increased or decrease in the melting temperature of DNA ? Why is this?

A

A high salt concetration leads to a higher melting temperature of DNA

This is because;

  • negative ions neutralise the repulsive negative phosphate groups on the backbone
  • the salt makes negatively charged phpshates and exisitng repulsion between phosphates leading to a higher DNA melting temperature
67
Q

How is DNA packaged into the nucleus?

A

The DNA is condensed in the form of chromatin and is then packaged into the nucleus

68
Q

What does chromatin contains and what forms does this come in, within the nucleus?

A

Chromatin contains 5 main proteins called Histones (histones wrap DNA around it and pack it into the nucleus).

The forms of histones are;

H1

H2A

H2B

H3

H4

69
Q

What do the histone proteins contain and what do they react with and why?

A

The histones are very basic and contains lots of positively charged amino acids such as; arginine and lysine

These interact with the negatively charged DNA (electrostatic attractions) due to the positive and negative charge

70
Q

What forms the structure of the histone core and what is it described as?

A

two copies of H2A, H2B, H3 and H4 combine to form an octameric, disk-like structure known as the histone core

71
Q

Whats a nucleosome?

A

When DNA is wrapped around a histone core (roughly 1.6 times)

72
Q

How many base pairs are found in the linking region between histone cores?

A

around 20 base pairs

73
Q

What can histone cores also help to regulate?

A

gene function

74
Q

Whats the role of the H1 histone?

A
  • linker histone
  • contracts both proteins and DNA
  • changes conformation of DNA as it leaves the nucleosome which helps to compact the DNA
75
Q

What were the three proposed theories for DNA replication and what was the idea within them?

A
  1. Conservative: maintains the double helix and theres one parent and one daughter
  2. Semi-conservative: parent strand provides a template for the daughter strand
  3. Dispersive: 50/50 mix of parent and daughter in each strand, alternating within the strand
76
Q

What was Meselson and Stahl’s experiment?

A

They grew bacteria on heavy (non-radioactive) isotope of nitrogen (15N): old DNA

cells containing labelled DNA were then transferred to a medium containing the normal isotop of nitrogen (14N): new DNA

The DNA was then isolated after 1, 2 and 3 generations

77
Q

What did each generation show in Meselson and Stahl’s experiment?

How was the DNA seperated?

A

At zero: all DNA has 15N

After generation 1: all DNA density was between 14N and 15N

After generation 2: 50% has density of 14N and 50% is intermediate

After generation 3: 75% has density 14N and 25% is intermediate

The DNA was seperated by density gradient centrifugation

78
Q

What happens in each of these centrifugations?

  1. Aside on centrifugation
  2. sucrose density centrifugation
  3. cesium chloride density gradient centrifugations
A

Aside on centrifugation:

  • the heavier particles sediment the fastest
  • not an equilibrium- everything goes to the bottom of the tube

Sucrose density centrifugation:

  • samples applies to the top of a tube that already contains a gradient of sucrose concentrations (more concentration at bottom)
  • not equilibrium method- everything goes to bottom of tube
  • gives better seperation of the fractions if stopped at right time

Cesium chloride density gradeint centrifugation

  • samples are dispersed in a concentrated solution of cesium chloride
  • this is an equilibrium method
  • under high centrifugal force the cesium forms a concentration gradient with the ions (denser at bottom of the tube)
  • seperates the samples in accordance to density, NOT mass of shape
  • DNA ‘floats’ in this position that corresponds to its buoyancy density
79
Q

Why is semi-conservative replication described as being bi-directional?

A

replication starts at a fixed point and then the replication occurs out from the origin

80
Q

What is the replication fork formed from and what is it’s resting structure?

A

The fork is formed from helicases and its resting strucutre has two branching ‘prongs’

81
Q

What direction is the synthesis in the replication fork?

A

in the 5’ to 3’ direction

82
Q

Tell me what is required for DNA replication?

A
  1. single stranded DNA template
  2. extended from an existing strand (primer)
  3. The primer to be made from RNA and made from the enzyme primase
83
Q

In the lagging strand of DNA, when made in short fragments in the 5’ –> 3’ directions, what are the fragments joined together by?

A

Okazaki fragments

84
Q

What’s the name of the enzyme in DNA that joins the short fragments together?

A

DNA ligase

85
Q

State two important uses of DNA polymerase reactions?

A
  1. DNA sequencing
  2. DNA amplification (PCR)
86
Q

What does DNA sequencing reactions contain?

A

All the usual dNTPs, plus a small amount of modified nucleotide triphosphate- ddNTP (dideoxynucleotide triphosphate)

87
Q

in DNA sequencing what has a lower concentration: ddNTP or dNTP?

A

lower amount of ddNTP compared to dNTPs

88
Q

If the DNA sequencing reactions contains dATP, dGTP, dTTP, dCTP and ddATP, what causes the synthesis to stop?

A

synthesis will stop everytime ddATP is incorporated

89
Q

How are DNA fragments seperated and what gel’s can be used?

A

DNA fragments are seperated via gel electrophesis.

the gels that can be used are;

  1. Agarose
  2. Polyacrylamide
90
Q

What type of gel is Agarose, what is it used for, whats used to visualise the DNA and what colour does it fluoresce

A

Agarose

  • a physical gel
  • used for seperating double stranded DNA fragments
  • horizontal submerged gel
  • DNA is visulaised by added a dye called ethidium which fluoresces orange
91
Q

What type of gel is Polyacrylamide, what is it mainly used for, what is the DNA visualised by?

A

Polyacrylamide

  • a chemical gel
  • used for denaturing gels containing 8M urea, to seperatre single-stranded DNA fragments
  • DNA is visualised by autoradiography (32P) or by covalernt attachment of a fluorescent group
92
Q

What’s next generation sequencing?

A
  • Short fragments of DNA as input, annealed to a slide using oligonucleotide adaptors
  • use of PCR to create multiple copies
  • use of fluorescent nucleotides for sequencing
  • use of a reversable terminator
93
Q

What are the steps to PCR?

A

1. denature the DNA to produce single strands using heat

2. add short primers that are complementary to the ends of the sequence of interest

Lower temperature –> anneal

3. Termostable DNA polymerase

  • isolated from Termus aquaticus (Taq) as it is heat stable so wont denature (however has a high error rate)
  • require for DNA extension

4. repeat steps of denaturation, annealing and extension

BIOL1007 (8 decks)

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