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YEAR 1 - Biochemistry and Medical Genetics (Anton) > 3. Molecular and Medical Genetics > Flashcards

3. Molecular and Medical Genetics Flashcards

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1
Q

Describe Gregor Mendel’s experiment and what conclusions it led to.

A

EXPERIMENT:

  • Focused on 7 main characteristics in pea plants.
  • Bred different homozygous plants together (e.g. RR and rr), then bred their offsrping.
  • In first generation, the dominant characteristic disappeared, but in the second generation it returned in the 3:1 ratio.
  • The 7 characteristics are independent of each of each other.

CONCLUSIONS:

  • Idea of alleles created.
  • Alleles segregate seperately.
  • Two different traits segregate independently of each other.
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2
Q

Define a gene.

A

An inherited section of DNA specifying phenotype at a gross or molecular level.

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3
Q

Describe the two levels at which a gene can have an effect on phenotype.

A
  • Gross level -> e.g. Morphological characteristics
  • Molecular level -> e.g. Products such as particular proteins
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4
Q

Describe the structure of a METAPHASE chromosome.

A

Each chromatid:

  • Telomere at both ends
  • Short arm (p)
  • Long arm (q)

Two identical chromatids are joined by a centromere.

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5
Q

What is the section joining two chromatids called?

A

Centromere

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6
Q

What are the short and long sections of a chromatid called?

A
  • Short - p (for “petit”)
  • Long - q
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7
Q

What are the short and long arms of a chromatid useful for?

A

Orientating a chromosome.

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8
Q

What are the different types of metaphase chromosome, based on their structure?

A

With the short (p) arm at the top:

  • Acrocentric - Very high-up centromere (acro = hill)
  • Submetacentric - Slightly high-up centromere
  • Metacentric - Centromere roughly in the middle
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9
Q

What are telocentric chromosomes?

A

Chromosomes where the centromere is in the telomeres, BUT these are not found in humans.

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10
Q

Draw a diagram of the different metaphase chromosome types.

A
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11
Q

Which chromosomes are acrocentric?

A

13, 14, 15, 21, 22, Y

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12
Q

Which chromosomes are metacentric?

A

1, 3, 19, 20

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13
Q

What is a karyotype?

A

The specific set of chromosomes each species has (e.g. the number and size of chromosomes).

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14
Q

Describe the human karyotype.

A
  • 22 autosomal pairs of chromosomes
  • 1 pair of sex chromosomes
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15
Q

Do the largest chromosomes contain the most genes?

A

Not necessarily, but there is a general correlation.

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16
Q

Describe the parts of the cell cycle.

A
  • Mitosis -> Division

When not in mitosis, the cell is in interphase:

  • Gap phase 1 (G1) -> Cellular contents (except chromosomes) duplicated
  • Synthesis -> Chromosomes duplicated
  • Gap phase 2 (G2) -> Preparation for mitosis

The cell can exit the cell cycle from G1 to enter G0, where the cell is quiescent.

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17
Q

Is a cell always in the cell cycle?

A
  • No, it can enter G0 from G1 and become a quiescent cell.
  • It can then return to the cell cycle later.
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18
Q

Draw the cell cycle.

A
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19
Q

Are chromosomes clearly visible in interphase?

A

No, they are loosely arranged in the nucleus, but chromosome territories can be distinguished.

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20
Q

What are chromosomes?

A

Units that contain an organism’s DNA, associated with DNA-binding proteins. This creates a macromolecular structure.

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21
Q

Describe the basic structure of a DNA in a chromosome.

A
  • DNA is formed from 4 basic nucleotides, containing the bases adenine, thymine, guanine and cytosine.
  • DNA is double stranded and adopts a double helix structure.
  • Hydrogen bonds between G≡C and A=T hold the helix together.
  • DNA-binding proteins combine in the chromsome.
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22
Q

What is DNA (and RNA) a polymer of?

A

Alternating phosphates and sugar residues.

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23
Q

What is the basic unit of DNA or RNA?

A

Nucleotide

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24
Q

How many carbons do the sugars in DNA and RNA have?

A

5

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25
Q

What is the charge of the phosphate-sugar backbone in DNA?

A

Negative

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26
Q

What is more stable, DNA or RNA?

A

DNA

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27
Q

What are the sugars in DNA and RNA?

A
  • DNA -> Deoxyribose
  • RNA -> Ribose
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28
Q

How are the carbons on DNA and RNA nucleotides numbered?

A

Carbon-1 is the carbon with the base attached to it.

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29
Q

What is the difference in structure between deoxyribose and ribose?

A

On carbon-2:

  • Deoxyribose has a H
  • Ribose has an OH
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30
Q

Draw the difference in structure of deoxyribose and ribose.

A
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31
Q

Where on deoxyribose and ribose does the base attach?

A

On carbon-1, the carbon on the opposite side of the O to the “tail” of the sugar.

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32
Q

Which DNA base is replaced by which base in RNA?

A

Thymine in DNA is replaced by uracil in RNA.

T -> U

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33
Q

How can nitrogenous bases be classified?

A

They are split into two categories:

  • Purines -> 2 rings
  • Pyrimidines -> 1 ring
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34
Q

What are purines and what do they include?

A

Nitrogenous bases with 2 rings. They include:

  • Adenine
  • Guanine
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35
Q

What are pyrimidines and what do they include?

A

Nitrogenous bases with 1 ring. They include:

  • Cytosine
  • Thymine
  • Uracil
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36
Q

What are a nucleoside and a nucleotide?

A
  • Nucleoside = Base + Sugar
  • Nucleotide = Base + Sugar + P
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37
Q

Where does a phosphate join onto a nucleoside to give a nucleotide?

A

It replaces the H from the OH on carbon-5 (on the tail), so that the P is surrounded by 4 O atoms.

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38
Q

What is the base, nucleoside and nucleotide for the A base?

A
  • Base = Adenine
  • Nucleoside = Adenosine
  • Nucleotide = e.g. AMP or dCTP
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39
Q

In nomenclature of nucleotides, how is it indicated that something is of deoxyribose derivation, not ribose?

A

A ‘d’ is added to the start of the shorthand name. For example, deoxyadenosine triphosphate is dATP.

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40
Q

Draw the structure of AMP.

A
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41
Q

Draw the structure of dCTP (deoxycytidine triphosphate).

A
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42
Q

Is DNA synthesised from deoxyribonucleoside monophosphates (dNMPs) or deoxyribonucleoside triphosphates (dNTPs)?

A

Deoxyribonucleoside triphosphates (dNTPs) -> The two phosphates that are not in the phosphodiester bond are cleaved off.

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43
Q

Between which carbons are nucleotides joined?

A

3I and 5I

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44
Q

Describe the process and mechanism by which two nucleotides may be joined to produce a nucleic acid chain.

A

DNA (or RNA) polymerases catalyse this:

  • OH on the 3-prime carbon carries out a nucleophilic attack on the phosphate closest to the sugar of a different nucleotide (remember: there are 3 phosphates attached)
  • The other 2 phosphates are lost as a molecule of pyrophosphate
  • A phosphodiester bond has been formed, which looks like this: C-O-P-O-C
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45
Q

What is formed when nucleotides are joined?

A

Oligonucleotides, then polynucleotides.

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46
Q

What is at the 3-prime and 5-prime end of a DNA strand?

A
  • 3-prime -> OH
  • 5-prime -> Phosphate
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47
Q

Draw the formation of a phosphodiester bond.

A
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48
Q

In which direction is DNA synthesised and why?

A

5-prime to 3-prime direction, because polymerase only works in 1 direction.

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49
Q

What is it important to remember about the charge of phosphate in DNA and RNA?

A

It is negative, which can create dipoles.

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50
Q

How many hydrogen bonds are formed between A and T as well as G and C?

A
  • A and T -> 2 bonds
  • G and C -> 3 bonds
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51
Q

Draw the formation of DNA hydrogen bonds.

A
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52
Q

What can be said about the direction of the two strands in a DNA helix?

A

They are anti-parallel.

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53
Q

What can single-stranded nucleic acid do?

A

Act as templates for transcription and replication.

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54
Q

What is an important feature to remember about the DNA double helix?

A

The double helix has major and minor grooves, which are important for molecules, such as transcription factors, to associate with the DNA.

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55
Q

Draw a diagram of a DNA double helix showing the different grooves.

A
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56
Q

What is chromatin?

A

The mass of genetic material composed of DNA and proteins that condense to form chromosomes during eukaryotic cell division.

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57
Q

Briefly describe how DNA is arranged in the nucleus.

A

DNA -> Chromatin -> Territories

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58
Q

What is the basic unit of chromatin?

A

Nucleosome

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59
Q

What is a nucleosome? Describe its structure.

A

It is the fundamental unit of DNA packing in a chromosome. It consists of:

  • A section of DNA (about 160 nucleotides)
  • Wrapped twice around 8 histone proteins
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60
Q

What are the histones in a nucleosome?

A

2 times: H2A, H2B, H3, H4

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61
Q

In detail, describe the way in which DNA is arranged into territories in the nucleus?

A
  • Sections of 160 nucleotides are wrapped twice around a histone
  • 8 histones are in a nucleosome
  • Nucleosomes are joined by linker DNA
  • Association of non-histone proteins creates chromatin
  • 3D architecture -> Caused by organisation into complex dynamic structures (such as chromatin loops that rae formed by ring-shaped proteins such as cohesin
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62
Q

Describe the levels of DNA structure.

A

PRIMARY

  • DNA double helix -> DNA sequence, methylation, genes

SECONDARY

  • Nucleosomes -> Nucleosome positioning, epigenetic modification, Chromatin, Accessibility

3D STRUCTURE

  1. Chromatin loops -> Promoter enhancer interactions
  2. TADs (Topologically Associated Domains)
  3. A/B compartments -> Chromatin scale: A = Active, B = Inactive
  4. Chromosome territory -> Nuclear space occupied
  5. Nucleus -> Relative positioning of chromosome territories
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63
Q

Check whether DNA has a dipole.

A

Do it.

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64
Q

What term can be used to describe DNA replication?

A

Semi-conservative

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65
Q

Describe simply the process of semi-conservative DNA replication.

A

The two antiparallel strands must be unwound and each may be used as a template for the synthesis of new complementary strands by dNTPs (nucleoside triphosphates) and polymerases.

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66
Q

During DNA replication, at what point is the double-helix opened?

A

At origins of replication.

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67
Q

What causes the DNA strands to open before DNA synthesis?

A

DNA helicases

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68
Q

What assembles at origins of replication and when?

A
  • Pre-replication complex
  • In the gap phase of the cell cycle (G1)
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69
Q

What allows DNA replication to start?

A

RNA primers are synthesised by primase on both strands.

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70
Q

In DNA replication, once the two strands are opened, what stops them from reannealing?

A
  • Single strand specific binding protein: SSB
  • For example, Replication protein A (RPA)
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71
Q

Describe how DNA replication is started.

A
  • In the gap phase of the cell cycle, a pre-replication complex is formed at the origins of replication.
  • DNA helicase moves to origins of replication, breaks open the hydrogen bonds and opens up the double helix. This process requires energy from ATP.
  • DNA polymerase requires an RNA primer in order to begin replication, which is a strand of RNA about 10-60 nucleotides long.
  • A primer is required on both strands of the DNA, so each strand can act as a template.
  • Replication protein A (RPA) binds single-stranded DNA and prevents reannealing.
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72
Q

How many origins of replication are there in human and bacterial DNA?

A
  • Human -> Multiple, which allows DNA to be transcribed at multiple points simultaneously.
  • Bacterial -> Just one.
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73
Q

What are the two types of new strand in DNA replication?

A
  • Leading
  • Lagging
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74
Q

What DNA replication starts, how many replication forks are there?

A

2 (one is created by DNA being synthesised on each strand in antiparallel directions)

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75
Q

Describe the concept of the leading and lagging strands in DNA replication.

A
  • As the DNA polymerases travel in antiparallel directions on each of the two strands, a fork is created in each direction.
  • This means that behind the leading strand there is a gap where only the opposite strand’s leading strand is forming.
  • This gap is filled by lagging strands that are continuously synthesised starting with new RNA primers.
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76
Q

What are Okazaki fragments?

A

Short sequences of DNA nucleotides which are synthesized discontinuously and later linked together to create the lagging strand during DNA replication.

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77
Q

In DNA replication, what do helicases do?

A

Unwind the DNA, using energy provided by hydrolysis of ATP.

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78
Q

In DNA replication, what does RPA stand for and what does it do?

A
  • Replication protein A
  • Binds to single-stranded DNA and prevents refolding
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79
Q

In DNA replication, what do topoisomerases do?

A

Reduce the torsional strain on DNA caused by unwinding.

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80
Q

What are the two types of DNA polymerase in DNA replication?

A
  • DNA polymerase α (a.k.a. Primase) -> Synthesises the RNA primer
  • DNA polymerase δ -> Synthesises the DNA strand
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81
Q

In DNA replication, what is PCNA and what does it do?

A
  • Clamp
  • Tethers DNA polymerase δ to DNA, displacing the primase.
  • Enables polymerase δ to be highly processive and synthesis can occur in long segments.
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82
Q

In DNA replication, what does RFC do?

A

Loads the PCNA “clamp” onto DNA.

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83
Q

In DNA replication, what does RNASe H do?

A

Degrades and removes the RNA primer.

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84
Q

Describe how lagging strand synthesis is ended in DNA replication.

A
  • When the lagging strand reaches the leading strand the DNA polymerase is displaced with the DNA helicase.
  • Okazaki fragments are joined when the DNA polymerase and helicase push aside the primer at the end of the next Okazaki strand, and FEN1 cuts the DNA just past the primer.
  • The remaining DNA is synthesised as the primer is removed and then the fragments are joined by DNA ligase.
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85
Q

In DNA replication, what does FEN1 do?

A

Removes RNA flap when Okazaki fragments are completed.

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86
Q

In DNA replication, what does ligase do?

A

Fuses the completed Okazaki fragments together in the lagging strand.

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87
Q

What is the function of telomeres?

A
  • Prevent shortening of chromosomes during proliferation
  • Enable correcting matching of the two DNA strands -> Stop end to end fusions of the DNA strands
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88
Q

What tandem repeat is found in telomeres?

A

TTAGGG

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89
Q

What happens to telomeres over time and why?

A
  • They get shorter with each replication, until cell apoptosis happens when they are too short
  • This is because there is no template for primase available to synthesis the next RNA primer for the last Okazaki fragment
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90
Q

What does telomerase do?

A

Extends telomeres, so they are not shortened too quickly.

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91
Q

What is unusual about the structure of telomerase?

A

It has an RNA component.

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92
Q

How does telomerase work?

A
  • It is a reverse transcriptase (TERT)
  • It uses an RNA template to synthesise the complementary DNA strand, which extends the telomeres
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93
Q

What is the clinical relevance of telomerase?

A

Overactivity of telomerase can lead to cell immortality in cancer cells.

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94
Q

What are the main mechanisms for repair of errors during DNA replication?

A
  • 3’ to 5’ exonucleolytic proofreading by the DNA polymerase
  • Non-polymerase mismatch repair
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95
Q

How common are errors in DNA polymerisation during synthesis?

A

1 in 105 nucleotides

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96
Q

What property of DNA polymerase allows it to repair its mistakes?

A

3’ to 5’ exonuclease activity

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97
Q

Does DNA polymerase have 3’ to 5’ or 5’ to 3’ exonuclease activity?

A

3’ to 5’ (this is opposite to the direction of DNA synthesis)

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98
Q

Describe how DNA polymerase 3’ to 5’ exonculease activity works.

A
  • If a wrong nucleotide is selected and inserted, base-pairing will be disrupted, causing a shift from the polymerase activity to the exo-activity.
  • The 3’ to 5’ exoribonuclease activity enables the polymerase to remove the ‘wrong’ nucleotide.
  • Polymerisation then resumes.
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99
Q

How common are errors in 3’ to 5’ exonuclease activity of DNA polymerase?

A

1 in 102 nucleotides

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100
Q

What are non-polymerase mismatch repair mechanisms for correcting errors in DNA synthesis and how do they work? What is the error rate?

A
  • They are mechanisms that recognise the newly-synthesised strand after replication (the mechanism for this in most species is not fully understood) and excise the section with the mismatch, before synthesising DNA to fill the gap.
  • Error rate: 1 in 102 nucleotides
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101
Q

After accounting for DNA repair mechanisms, how common are errors in DNA replication?

A
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102
Q

What happens to histones in DNA replication?

A
  • Histones are removed when the replication fork moves forward.
  • In the daughter strands, one half of each histone is recycled from the parental, the other is newly synthesised.
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103
Q

What are the two types of mutation?

A
  • Spontaneous
    • Changes in chemical properties of the nucleic acids
    • Problems during replication or cell division
  • Induced
    • Radiation
    • Chemicals
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104
Q

What are two examples of mutations caused by changes to the chemical properties of nucleotides?

A
  • Deamination
  • Depurination
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105
Q

What is deamination of nucleotides and what is the consequence?

A
  • It is removal of an amino group from a nucleotide
  • This can cause the type of nucleotide to be changed (e.g. from C to U)
  • The result is that the nucleotide sequence is altered in ONE of the daughter molecules in DNA replication
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106
Q

Which nucleotide in particular may be affected by deamination?

A

Cytosine can be converted to uracil.

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107
Q

What is depurination of nucleotides and what is the consequence?

A
  • It is removal of the purine base from a nucleotide
  • This leaves just the sugar-phosphate backbone in that part of the DNA strand, which is effectively a deletion
  • The result is that the nucleotide sequence is altered by frame shift in ONE of the daughter molecules in DNA replication
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108
Q

What are two examples of mutations that can occur during DNA replication?

A
  • Base mismatch (e.g. an A pairing with a C)
  • Polymerase slippage
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109
Q

What is the result of polymerase slippage during DNA replication?

A

A repeat region can be extended.

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110
Q

Give an example of radiation causing mutations.

A
  • Exposure to UV can cause dimerisation of thymine.
  • Two thymines are covalently linked -> Affects the structure of the DNA -> Affects replication.
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111
Q

What are the different repair systems for DNA mutations?

A
  • Direct repair
  • Excision repair
  • Mismatch repair
  • Nonhomologous end-joining
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112
Q

What is the direct repair mechanism of DNA repair and when is it used?

A
  • Single nucleotides that have been damaged by transfer of methyl/ethyl group onto the base (e.g. by alkylating agents) are repaired this way
  • This is done by transferring the alkyl group onto the enzyme
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113
Q

What is the excision repair mechanism of DNA repair and when is it used?

A
  • Used to repair cytosine nucleotides that have been deaminated to give uracil
  • This involves single nucleotide excision repair DNA glycosylases that cut the nucleotide out and resynthesise the correct DNA
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114
Q

What is the mismatch repair mechanism of DNA repair and when is it used?

A
  • The mismatched nucleotide is cut out and the correct DNA is resynthesised
  • Thymidine dimers require excision of a larger area
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115
Q

What are homologous and non-homologous recombination and when are they used? What is the difference between them?

A
  • They are methods of repairing DNA molecules that have a double-strand break in them
  • Non-homologous involves just joining the strands back up, while homologous reocmbination involve creating sort of sticky ends (CHECK THIS)
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116
Q

What are the different types of mutation and how is each repaired?

A
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117
Q

What are some examples of diseases caused by mutations in DNA repair genes? [EXTRA?]

A
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118
Q

What is a missense mutation?

A

A mutation that alters a codon so that it is recognised by a different tRNA and consequently a different amino-acid is introduced into the polypeptide chain.

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119
Q

What is a nonsense mutation?

A
  • A change that introduces a premature stop codon in the mRNA.
  • This results in the production of a shortened protein that might be nonfunctional or displays an altered function or regulation
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120
Q

What are the different types of substitution mutation and what is the effect on the protein?

A
  • Synonymous
    • Silent -> No effect on protein
  • Non-synonymous
    • Missense -> Altered amino acid
    • Nonsense -> Stop codon produced
    • Splicing -> Various
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121
Q

What are the different types of deletion mutation and what is the effect on the protein?

A
  • Multiple of 3 -> Various effects
  • Not a multiple of 3
    • Frame shift -> Gain/Loss of function, Premature termination
  • Large deletion
    • Partial or whole gene deletion -> Loss of function/expression
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122
Q

What are the different types of insertion mutation and what is the effect on the protein?

A
  • Multiple of 3 -> Various effects
  • Not a multiple of 3
    • Frame shift -> Gain/Loss of function, Premature termination
  • Large deletion
    • Partial or whole gene duplication -> May affect dosage
  • Trinucleotide
    • Dynamic mutation -> Altered function, stability
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123
Q

What does homozygous mean?

A

When an organism has two copies of the same allele for a given gene.

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124
Q

What does heterozygous mean?

A

When an organism has two different alleles for a given gene.

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125
Q

Write an ‘equation’ to show how phenotype and genotype are related/

A

Phenotype = Genotype + Environment

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126
Q

What are the two genomes in a eukaryotic cell?

A
  • Nuclear genome
  • Mitochondrial genome
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127
Q

Compare the structure and inheritance pattern of the nuclear and mitochondrial genome.

A

Nuclear:

  • Linear chromosomes
  • Maternal and paternal inheritance

Mitochondrial:

  • Circular chromosomes
  • Maternal inheritance only
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128
Q

Compare the different sequence types in the nuclear and mitochondrial genomes.

A
  • Nuclear genome features mostly poorly conserved sequences
  • Mitochondrial genome features mostly highly conserved sequences
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129
Q

How many genes does the mitochondrial genome contain? How many of these code for polypeptides and how many code for RNA?

A

37 genes:

  • 13 encode polypeptides
  • 24 encode non-coding RNAs (22 t-RNAs and 2 rRNAs)
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130
Q

Does the mitochondrial genome contain introns?

A

No

131
Q

Is the majority of the mitochondrial proteome encoded in the mitochondrial or nuclear genome?

A

Nuclear -> The polypeptides are then imported into the mitochondria.

132
Q

Compare the nuclear and mitochondrial genome in terms of:

  • Number of protein-coding genes
  • Number of RNA genes
  • Gene density
  • Transcription
  • Introns
  • Percentage of protein-coding DNA
A
133
Q

What percentage of the human genome contains repeat regions?

A

40%

134
Q

Describe the different types of repeat regions in the human genome. [EXTRA?]

A
135
Q

Draw the central dogma of gene expression, including the cellular compartments where each part happens.

A
136
Q

What comes first, transcription or translation?

A

Transcription

137
Q

Which enzyme transcribes DNA into RNA?

A

RNA polymerase

138
Q

Draw a diagram to show how RNA polymerase transcribes DNA.

A
139
Q

What are the two strands of DNA called during transcription?

A
  • Template strand -> The one that free nucleotides bind to and is transcribed
  • Coding strand -> The one that is not transcribed, but the base order matches the RNA strand
140
Q

When transcribing DNA into RNA, which base replaces which?

A

Thymine (T) is replaced by uracil (U)

141
Q

Can each strand in the DNA molecule be used as the template strand?

A

Yes, the RNA polymerase can use both the top and bottom strand as the template strand.

142
Q

In which direction is a DNA strand read during transcription and in which direction is the RNA strand synthesised?

A
  • DNA strand reading -> 3’ to 5’ direction
  • RNA strand synthesis -> 5’ to 3’ direction
143
Q

Do RNA polymerases require a primer?

A

No, unlike DNA polymerases they do not.

144
Q

H|ow many types of RNA polymerase are there in eukaryotes?

A

3

145
Q

What does RNA polymerase I transcribe? [IMPORTANT]

A

rRNA

146
Q

What does RNA polymerase II transcribe? [IMPORTANT]

A

mRNA, snRNA, miRNA

147
Q

What does RNA polymerase III transcribe? [IMPORTANT]

A

tRNA, some snRNA

148
Q

What does each of the three eukaryotic RNA polymerases transcribe?

A
149
Q

What is the function of mRNAs?

A

Code for proteins

150
Q

What is the function of rRNAs?

A

Form the core of ribosomes and catalyse the formation of proteins.

151
Q

What is the function of miRNAs?

A

Regulate gene expression.

152
Q

What is the function of tRNAs?

A

Involved in converting from mRNA to amino acid chain during protein synthesis.

153
Q

What are the functions of other small RNAs (aside from mRNAs, rRNAs, tRNAs and miRNAs)?

A

Used in:

  • RNA splicing
  • Telomere maintenance
  • Other processes
154
Q

Draw a summary of the roles of different types of eukaryotic RNA.

A
155
Q

How do RNA polymerases know where to start transcribing?

A

They bind to promoter sequences on the DNA.

156
Q

What do prokaryotes and eukaryotes require to start transcription aside from RNA polymerase?

A

Prokaryotes:

  • Sigma factors
  • Different numbers of sigma factors exist in different species and each type guides the polymerase to the promoter region for a different gene

Eukaryotes:

  • General transcription factors (GTFs).
  • Different combinations of transcription factor attract each of the three types of eukaryotic RNA polymerase by binding to the core promoter first
157
Q

Where are promoters found relative to a gene?

A

Upstream of the gene

158
Q

In eukaryotes, what recruits the polymerase to the promoter region?

A

General transcription factors (GTFs)

159
Q

What are some consensus sequences in prokaryotic promoter regions? [IMPORTANT]

A
  • Pribnow box at the -10 region, which is a TATAAT sequence where the DNA strands first separate (partly due to the small number of hydrogen bonds between A and T bases).
  • There is a second consensus sequence at the -35 region.
160
Q

How are eukaryotic promoter regions different from prokaryotic promoter regions?

A
  • Eukaryote promoters are more complex and include core, proximal and distal promoters.
  • Eukaryotes are also distinct in their downstream promoter regions (past the 3-prime end of the TSS).
161
Q

What are the different types of eukaryotic promoters? [IMPORTANT]

A
  • Core -> Required for transcription and contain the TSS (transcription start site)
  • Proximal and distal -> Regulatory role
162
Q

What is an example of a consensus sequence in the core promoter of eukaryotic genes?

A

TATA box

163
Q

Draw the structure of a eukaryotic promoter region. [IMPORTANT]

A

Note the core, proximal and distal promoters.

164
Q

How are transcription factors named?

A

They all start with TF.

e.g. TFIIA

165
Q

What is the transcription start site (TSS)?

A

It is the point just past the promoter region at which transcription of a gene starts.

166
Q

What are the different types of transcription factor in eukaryotes?

A
  • General transcription factors (GTFs) -> Bind to the core promoter and are required for transcription since they recruit the RNA polymerase
  • Activators and co-activators -> Bind to other regions on the DNA to regulate transcription of specific genes
167
Q

What do these regulatory regions do in eukaryotes:

  • Enhancer
  • Silencer
  • Insulator
  • Locus control region
A
168
Q

Draw a diagram to summarise the control of transcription at different points in DNA.

A
169
Q

Describe the process of elongation (in transcription) in eukaryotes.

A
  • RNA polymerase moves along the template DNA strand in the 3-prime to 5-prime direction (polymerase can only synthesise DNA in the 5-prime to 3-prime direction).
  • The polymerase separates the two DNA strands, forming an open complex where free RNA nucleotides can join to the template strand by complementary base pairing. This region is called the transcription bubble.
  • Phosphodiester bonds are synthesised between the NTPs
  • Once synthesised, the growing RNA chain does not remain associated with the DNA strand (as in DNA replication), but is removed through an RNA exit channel.
170
Q

Describe and draw the formation of a phosphodiester bond.

A
  • Phosphodiester bonds are synthesised between the NTPs when the -OH on the 3-prime end of the previous NTP carries out a nucleophilic attack on the first phosphate (alpha phosphate) of the next NTP.
  • This releases a pyrophosphate molecule.
171
Q

Compare control of transcription in prokaryotes and eukaryotes. [IMPORTANT]

A

Prokaryotes:

  • Genes are set by default to “on”, so that without any regulatory molecules there will be some degree of transcription.
  • Therefore, the most basic form is negative control.
  • Prokaryotes feature both positive and negative control in structures called operons, which are sets of (usually functionally related) genes that are under the control of a single operator within a regulatory region.
  • The operon contains the genes to be transcribed, the operator and the promoter (where the RNA polymerase binds).
  • Negative control is due to repressors binding to the operator, while positive control is due to activators binding to a different site within the regulatory region, which increases the affinity of polymerase for the promoter.

Eukaryotes:

  • Genes are by default set to “off” and require a set of transcription factors in order to initiate transcription.
  • This gives the opportunity for control pathways that affect the general transcription factors.
  • Aside from this, eukaryotic gene expression can be influenced across a larger distance, namely by enhancer and silencer regions that activate and repress transcription from regions outside of the promoter.
  • In eukaryotes, another level of gene regulation is added by the presence of histones, which can be enzymatically modified by acetylation or methylation.
172
Q

In prokaryotes, are genes set to on or off by default?

A

On

173
Q

What is an operon and what does it contain? How does it work?

A
  • Operons are sets of (usually functionally related) genes that are under the control of a single operator within a regulatory region.
  • Found in PROKARYOTES.
  • The operon contains:
    • Genes to be transcribed
    • Operator
    • Promoter (where the RNA polymerase binds).
  • Negative control is due to repressors binding to the operator, while positive control is due to activators binding to a different site within the regulatory region, which increases the affinity of polymerase for the promoter.
174
Q

What enables long-range control of transcription in eukaryotes?

A

Folding of the DNA in, for example, loops, so that transcription factors bound at the distal regulatory regions can interact with the general transcription factors and RNA polymerase.

175
Q

What is the difference between heterochromatin and euchromatin? Which is transcribed more?

A
  • Heterochromatin -> Tightly wrapped DNA regions containing inactive genes
  • Euchromatin -> Loosely wrapped DNA regions containing active genes
176
Q

What are the two main types of modification that can be done to histones to modify transcription of genes?

A
  • Acetylation
  • Methylation
177
Q

How do acetylation and methylation of histones affect the rate of transcription of genes?

A
  • Acetylation -> Increases transcription
  • Methylation -> Decreases transcription

These are general effects, but they can vary.

178
Q

How does DNA methylation affect transcription and why?

A
  • It decreases the rate of transcription.
  • This is because it impedes the binding of transcriptional proteins to the gene and methylated DNA may be bound by transcription repressor proteins.
179
Q

Describe the structure of heterochromatin and how that affects the rate of transcription of genes.

A
  • Nucleosomes are tightly packed and associated with additional heterochromatin proteins that bind to the specifically modified histone tails of nucleosomes in those regions.
  • This means the DNA in these regions is inaccessible and prevents the expression of genes that are located within these regions.
180
Q

What are constitutive heterochromatin and where is it found?

A
  • It is DNA that remains in a condensed state throughout the cell cycle.
  • It contains repeat sequences which are not transcribed and plays a role in chromosome structure
  • Found in: Centromere + Telomeres + Microsatellites throughout the chromosome
181
Q

Draw a mechnism for X inactivation [EXTRA?].

A
182
Q

Describe the structure of euchromatin and how that affects the rate of transcription of genes.

A
  • Nucleosomes are more loosely packed.
  • This exposes regions that are accessible to regulatory proteins.
  • Level of accessibility of DNA can still vary greatly -> Modifications of the histone tails also regulate accessibility at this scale.
183
Q

Draw the different levels of control of transcription in eukaryotes.

A
184
Q

What causes the transition between the initiation phase and elongation phase of transcription in eukaryotes?

A
  • The largest subunit of RNA polymerase II has a characteristic structure at its C-terminal domain (CTD)
  • The CTD consists of 52 repeats of a seven aminoacids YSPTSPS.
  • Phosphorylation of the serine residues in the CTD is critical for the transition to elongation.
185
Q

What phosphorylates the CTD of RNA polymerase II, causing the transition from initiation to elongation of transcription?

A

TFIIH (A transcription factor)

186
Q

Draw the structure of a bacterial gene.

A
187
Q

Draw the structure of a eukaryotic gene.

A
188
Q

Do you need to know how transcription is terminated?

A

No, that is right-hand side content.

189
Q

What are introns and exons? What happens to each?

A

In eukaryotes, exons are parts of genes that are expressed, while introns are also transcribed, but they are they spliced out during pre-mRNA processing.

190
Q

What are the 3 main pre-mRNA modifications that happen after transcription?

A
  • Capping the 5’ end
  • Splicing of introns
  • 3’ end formation
191
Q

Does pre-mRNA processing (e.g. splicing) occur during or after transcription?

A

During -> It is a co-transcriptional process.

192
Q

How is RNA polymerase II related to pre-mRNA processing?

A
  • When phosphorylated, the CTD (C-terminal domain) of RNA polymerase II serves as a landing pad for the 3 types of processing factor
  • This means that pre-mRNA processing can occur alongside transcription
193
Q

What is the first pre-mRNA processing event to occur?

A

Capping of the 5’ end of the mRNA (the 5’ end is the end that is produced first)

194
Q

Describe the capping of the 5’ end of mRNA (the first pre-mRNA processing event).

A
  • A GMP nucleotide is added to the 5’ end (in an unusual 5’ to 5’ linkage)
  • The G is then methylated
  • A cap binding complex (CBC) then binds to the GMP nucleotide
195
Q

What is the function of capping the 5’ end of mRNA (the first pre-mRNA processing event)?

A

The cap:

  • Protects the mRNA from degradation by 5’ exoribonucleases.
  • Facilitates translation initiation.
196
Q

What is the second pre-mRNA processing event?

A

Splicing

197
Q

What type of reaction is splicing?

A

Trans-esterification

198
Q

Draw the mechanism for splicing.

A
199
Q

What defines where splice sites are?

A

Cis-elements (near the splice sites) define the starts and ends of DNA.

200
Q

What does snRNA stand for?

A

Small nuclear RNA

201
Q

What is a spliceosome?

A
  • A large and complex molecular machine found primarily within the nucleus of eukaryotic cells.
  • It is responsible for splicing.
202
Q

What is a spliceosome composed of?

A
  • 5 RNA components -> U1, U2, U3, U4 and U5 snRNAs
  • These are bound to specific proteins

Together, each U snRNA and bound proteins are referred to as snRNPs.

203
Q

What are snRNPs?

A
  • Small nuclear ribonucleic RNA proteins
  • These are the protein-RNA complexes that form the spliceosome required for splicing.
204
Q

What does the spliceosome bind to?

A

Cis-elements in the pre-mRNA.

205
Q

How does splicing affect the Open Reading Frame?

A

It creates an mRNA molecule with a continued Open Reading Frame (ORF).

206
Q

What is the point of having introns in DNA?

A

It allows for alternative splicing.

207
Q

What is the function of alternative splicing?

A
  • Allows permutation of exons, enabling cells to potentially generate several proteins from a single gene
  • Plays an important role in tissue specific gene expression
208
Q

Describe the principle by which alternative splicing can occur.

A

Proteins that associate with certain sequences on the pre-mRNA can strengthen or weaken splice sites and so regulate exclusion or inclusion of exons.

209
Q

Describe how an exon can be kept in the mRNA during splicing.

A
  • The association of enhancer proteins with the pre-mRNA makes it more likely that the splicing factors recognise the 3’ and 5’ splice sites that flank an exon.
  • This increases the likelihood of it being kept in the mRNA.
210
Q

Describe how an exon can be removed from mRNA during splicing.

A
  • Negative factors binding to specific sequences can prevent recognition of both the 3’ and 5’ splice sites that flank an exon and splicing will skip the exon.
  • This results in potential exclusion of the exon.
211
Q

What is the third pre-mRNA processing event?

A

3’ end processing

212
Q

What are the two main parts of 3’ end processing in pre-mRNA processing?

A
  • Cleavage
  • Polyadenylation
213
Q

Describe the cis elements (parts of the pre-mRNA) that signal cleavage and polyadenylation at the 3’ end during pre-mRNA processing.

A

It has two parts:

  • The major signal for both cleavage and polyadenylation is the hexamer AATAAA (AAUAAA) which is located about 30 bases upstream of the cleavage and polyadenylation site.
  • A second signal, the downstream sequence element (DSE) is GT (GU) rich and is found after the cleavage site.
214
Q

What hexamer is the signal in pre-mRNA for cleavage and polyadenylation at the 3’ end?

A

AAUAAA (In DNA it is: AATAAA)

215
Q

What is the DSE in pre-mRNA for cleavage and polyadenylation at the 3’ end?

A
  • Downstream sequence element
  • It is GU rich (In DNA, it is GT rich)
216
Q

Describe how cleavage and polyadenylation occur in pre-mRNA processing.

A
  • The cleavage and polyadenylation machinery associates with the AAUAAA and DSE sequences on the pre-mRNA.
  • Cleavage occurs and the 3’end of the mRNA is then polyadenylated by poly(A) polymerase PAP.
217
Q

What enzyme polyadenylates pre-mRNA at the 3’ end after cleavage?

A

Poly(A) polymerase

(a.k.a. PAP)

218
Q

How many adenosine residues are added during polyadenylation in pre-mRNA processing?

A

200-250

219
Q

What happens after polyadenylation in pre-mRNA processing?

A

The poly(A) tail is covered by poly(A) binding proteins (PABP).

220
Q

What is the importance of polyadenylation in pre-mRNA processing?

A

Polyadenylation creates a uniform 3’-end to all protein encoding mRNAs which is critical for nuclear cytoplasmic export, stability and translatability of the mRNA.

221
Q

Draw the structure of mature mRNA (after pre-mRNA processing).

A
222
Q

How can errors in pre-mRNA processing lead to disease?

A

Mutations that affect the splicing pattern and/or splicing efficiency of a particular exon are common.

223
Q

What things can cause errors in pre-mRNA processing, leading to disease?

A
  • Mutations affecting splice sites
  • Mutations affecting splicing factors
  • Mutations affecting poly(A) signals
224
Q

How can mutations in splice sites lead to disease?

A
  • Can lead to reduced splicing out of particular introns -> Leads to faulty mRNAs
  • Mutations affecting 3’ splice sites can result in exon skipping
225
Q

How can mutations in cleavage and polyadenylation signal sites lead to disease?

A
  • Mutations affecting poly(A) signals can reduce or increase cleavage and polyadenylation.
  • This affects the total output of mRNA.
226
Q

What is the basic unit of mRNA?

A

Codon (3 bases)

227
Q

Describe the relationship between how much mRNA codes for how much protein.

A

One trinucleotide codon codes for an amino acid.

228
Q

What is the start codon in mRNA?

A

AUG

229
Q

What is the first amino acid in all proteins? Why?

A

Methionine -> This is because the start codon (AUG) codes for methionine.

230
Q

What are the 3 main stop codons?

A
  • UAA
  • UAG
  • UGA
231
Q

How do stop codons lead to termination of translation?

A

They are recognised by proteins instead of tRNA molecules, so this forces the termination of translation.

232
Q

How many reading frames does mRNA have?

A

3

233
Q

What is an open reading frame?

A

The section of mRNA from the start codon that includes all of the codons that code for amino acids.

234
Q

What type of mutation causes beta thalassemia?

A

A point mutation that produces a premature stop codon -> Nonsense mutation

235
Q

What type of mutation causes sickle-cell anaemia?

A

Missense mutation -> Where an amino acid is replaced by another.

236
Q

What things are required for translation?

A
  • tRNAs
  • Ribosomes
  • Translation factors
  • Energy (in the form of GTP)
237
Q

What assembles tRNA molecules?

A

Specific aminoacyl tRNA synthetases

238
Q

Draw the structure of a tRNA molecule.

A
239
Q

What do aminoacyl tRNA synthetases do and what is required for their action?

A
  • Charge t-RNA molecules with the correct amino-acids
  • This requires energy from ATP
240
Q

What ensures a high accuracy of aminoacyl tRNA synthetase activity?

A

Their action is highly specific and “proof reading” ensures accuracy of this process.

241
Q

What are the two components of ribosomes?

A
  • RNA
  • Proteins
242
Q

In ribosomes, is it the RNA or protein that has the catalytic component?

A

RNA is the catalytic component: Ribozyme

243
Q

In eukaryotes, where are ribosomes made?

A

In the nucleolus and then they are exported.

244
Q

What is the name for the eukaryotic and prokaryotic ribosome?

A
  • Eukaryotic -> 80S
  • Prokaryotic -> 70S
245
Q

Draw the structure of a prokaryotic ribosome.

A
246
Q

Draw the structure of a eukaryotic ribosome.

A
247
Q

What is the name for the three bases on a tRNA molecule?

A

Anticodon

248
Q

How is mRNA decoded by tRNAs?

A

The tRNA recognises its corresponding codon in the mRNA via base pairing between nucleotides in the anticodon and the codon.

249
Q

What is the consequence of genetic code being degenerate?

A

Some tRNA molecules can have more than one version to recognise more than one codon (e.g. there can be an Ala1 and Ala2 tRNA for alanine), and each of these may be able to recognise more than one codon.

250
Q

What is the wobble position and why is it important?

A
  • It is the third base in a codon
  • Base paring rules at the third base (wobble position) are less strict and a base can pair with more than one complementary base
251
Q

Draw the mechanism for elongation in translation. [EXTRA?]

A
252
Q

In which direction is mRNA read?

A

5’ to 3’ (opposite direction to DNA)

253
Q

During elongation in translation, to which end of the previous amino acid is the next amino acid added?

A

The C terminus.

254
Q

In which subunit of eukaryotic ribosomes are there 3 important sites for translation?

A

60S

255
Q

What are the three tRNA binding sites in the 60S subunit of eukaryotic ribosomes? How do they allow for polypeptide synthesis?

A
  • E site -> Exit site
  • P site -> Peptidyl site
  • A site -> Aminoacyl site
256
Q

When a tRNA molecule goes to a ribosome, where does it bind first?

A

It goes to the aminoacyl (A) site of the 60S subunit.

257
Q

What enzyme (of the ribosome) catalyses peptide bond formation during translation?

A

Peptidyl transferase

258
Q

How does a stop codon affect a ribosome?

A

It causes release of the peptide and dissociation of the ribosome.

259
Q

On which end of mRNA is the poly(A) tail and on which end is the cap?

A
  • Poly(A) tail -> 3’ end
  • Cap -> 5’ end
260
Q

What is the role of the 3’ poly(A) tail and 5’ cap in translation?

A
  • Cap binding proteins and proteins that bind the poly(A) tail interact with each other and create a circular mRNA molecule.
  • The small ribosomal subunit is recruited to the cap and then scans the mRNA for the AUG start codon.
  • Once the start codon is identified, the large ribosomal subunit joins and translation can commence.
261
Q

What are the two main regulation point in translation? [EXTRA]

A
  • Phosphorylation of translation initiation factors
  • Sequences that are present in the mRNA template
262
Q

Give an example of regulation of translation via phosphorylation of translation initiation factors.

A
263
Q

What are UTRs in mRNA and why are they important?

A
  • Untranslated regions
  • They are the bits of mRNA on either side of the open reading frame
  • They contain many regulatory sequences that regulate translation
264
Q

Draw how iron levels can affect translation. [EXTRA?]

A
265
Q

Describe how splicing allows for protection against synthesis of mutated proteins.

A
  • After splicing, EJC (exon junction complex) protein assemble at the junctions between exons
  • These serve as markers
  • The presence of an EJC downstream of a stop codon identifies the mRNA as faulty and induces its destruction (degradation).
266
Q

What are EJC proteins?

A

Exon junction complex proteins -> These are protein complexes that assemble at junction between exons after splicing of mRNA. They serve as markers.

267
Q

Draw a summary of the different stages at which protein expression can be regulated.

A
268
Q

Name some antibiotics that act by inhibiting DNA replication. [EXTRA?]

A

Quinolones

269
Q

Name some antibiotics that act by inhibiting transcription. [EXTRA?]

A

Rifamycin

270
Q

Name some antibiotics that act by inhibiting translation. [EXTRA?]

A
  • Tetracycline
  • Erythromycin
  • Streptomycin
  • Chloramphenicol
271
Q

How do these antibiotics work:

  • Aminoglycosides
  • Tetracycline
  • Chloramphenicol
  • Macrolide

[EXTRA?]

A
272
Q

What are the main categories of techniques used in the analysis of gene expression/gene function?

A
  • Hybridisation techniques
  • Enzyme-based techniques
  • Antibody techniques
273
Q

What are the main hybridisation techniques that you need to know about?

A
  • Southern blotting
  • Northern blotting
  • Fluorescent in situ hybridisation (FISH)
274
Q

What are the main enzyme-based techniques (to study genes and gene expression) that you need to know about?

A
  • PCR
  • RT-PCR
  • DNA sequencing
  • NGS
  • Manipulation of DNA -> Restriction, ligation, cloning
275
Q

What are the main antibody-based techniques (to study genes and gene expression) that you need to know about?

A
  • Western blotting
  • Elisa
  • Imaging
  • Immunoprecipitation
276
Q

How does heat affect the hybridisation of nucleic acids?

A
  • Double stranded molecules can be denatured (separated) by heat
  • Complementary sequences re-nature (re-hybridise) at lower temperature
277
Q

What are Northern, Western and Southern blotting?

A
  • Northern blotting -> Detection of the presence of specific RNA molecules in a sample
  • Western blotting -> Detection of the presence of specific proteins in a sample
  • Southern blotting -> Detection of the presence of specific DNA molecules in a sample
278
Q

Describe how Northern, Western and Southern blotting work.

A
  • Target molecules (e.g. RNA, proteins or DNA) are isolated -> DNA is usually fragmented
  • Separated by size using gel electrophoresis
  • Blotted onto a membrane, so that probes or antibodies can access them
  • Probes and antibodies are chosen to be specific to a certain RNA, DNA or protein
  • In Northern blotting, a hybridisation probe is used, which is an RNA or DNA sequence that is complementary to the target RNA sequence and is labelled fluorescently or radioactively
  • In Western blotting, an antibody is used that is specific for the target protein and is labelled or can be bound to by a labelled secondary antibody
  • In Southern blotting, a hybridisation probe is used, which is an RNA or DNA sequence that is complementary to the target DNA sequence and is labelled fluorescently or radioactively
  • Excess antibodies or antibodies are washed away
  • Labels are detected -> e.g. Using a X-ray film for radioactive probes -> This shows whether the target molecule is present
279
Q

What does Northern blotting detect?

A

The presence of a specific RNA sequence.

280
Q

What does Southern blotting detect?

A

The presence of a specific DNA in a sample.

281
Q

What does Western blotting detect?

A

The presence of a specific protein in a sample.

282
Q

What is a good mnemonic for remembering the blotting techniques?

A

S-D -> Southern-DNA
N-R -> Northern-RNA
O-O -> Nothing
W-P -> Western-Protein

283
Q

Describe what FISH is and how it works.

A

Fluorescent in situ hybridisation:

  • Uses fluorescent probes that bind to only complementary nucleic acid sequences.
  • Used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
  • Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes.
284
Q

How is FISH different from Southern blotting?

A

FISH is done within the cell (in situ), while Southern blotting involves some processing of the DNA before the probes are added. [CHECK THIS]

285
Q

What is chromosome painting and why is it useful?

A
  • The use of FISH to colour entire choromosomes.
  • This can be useful in characterising chromosomal abnormalities.
286
Q

What is RNA interference and how can it be exploited clinically?

A
  • Where cells use small ~21 nucleotide long RNAs (e.g. miRNAs) to regulate the expression of genes.
  • The small RNAs are incorporated into the RNA induced silencing complex (RISC) and act as guides for RISC to interact in a sequence specific manner with the target mRNAs.
  • RISC can then degrade the target mRNA or inhibit its translation.

This can be used to clinically knock out certain genes.

287
Q

What is the CRISPR/Cas9 system and what is it used for?

A

It is a microbial adaptive immune system that has been repurposed as gene editing system that can be used to generate or correct sequence changes in DNA.

288
Q

What does CRISPR/Cas9 mean?

A

CRISPR (clustered regulatory interspaced short palindromic sequences) associated Ca9 nuclease

289
Q

Describe briefly how CRISPR/Cas9 works.

A
  • The Cas9 nuclease can be introduced into eukaryotic cells and using guide RNAs specifically directed to any location in the genome where it can cut the DNA, leading to a break.
  • When the DNA tries to repair itself, two things can happen:
    • Homologous end joining -> This happens when a homologous sequence is provided along with the Cas9 nuclease, so that it acts as a template and therefore the DNA in the cell is edited to whatever is desired
    • Non-homologous end joining -> This happens when no homologous sequence is provided, so that the DNA either repairs itslef to be like before or it repairs itself by excising bases, leading to a non-functional protein
290
Q

Give some examples of the changes that the CRISPR/Cas9 system can produce in DNA.

A
291
Q

What sequences do restriction endonucleases typically cut at?

A

Palindromic 6 base sequences

292
Q

What is a restriction fragment length polymorphism? How can it be detected?

A
  • It is when there is a mutation in the recognition site of restriction endonuclease.
  • These can be detected by addition of restriction endonucleases and then separation of the fragments by electrophoresis.
  • This is because a mutation causes changes in lengths of the fragments.
293
Q

How can repeat expansion mutations be detected by restriction endonucleases?

A
  • The restriction endonucleases can be used to create fragments
  • These fragments can be separated by electrophoresis
  • Differences in their length from normal indicate mutations
294
Q

What is electrophoresis used for?

A

Separation of DNA fragments by their size.

295
Q

What does ligase do?

A

It can join DNA fragments that have been cut by restriction endonucleases.

296
Q

What is molecular cloning and what is it used for?

A
  • Forming recombinant DNA molecules, then replicating them within a host organism
  • Used to obtain multiple copies of a desired section of DNA within a cell
297
Q

Describe how molecular cloning works.

A
  • Vector is selected -> Usually a bacterial plasmid that contains an origin of replication, drug-resistance gene, and a recognition site for restriction endonucleases
  • Same restriction endonuclease used to cut target DNA and bacterial plasmid
  • Restriction endonucleases cut DNA at palindromic recognition sites by, leaving sticky or blunt end fragments
  • Recombinant DNA is formed by joining the vector DNA with the target DNA using DNA ligase, which forms phosphodiester bonds.
  • Recombinant DNA is placed in the host cells (transformation)
  • Methods for increasing competency include:
    • Placing the bacterial cells in cold calcium chloride, then exposing then to a brief heat shock at around 42°C
    • Electroporation -> Exposing cells to an electric field, which temporarily forms holes in the cell membrane
  • Cells are placed in agar that contains an antibiotic -> Cells which are successfully transformed contain the plasmid with the gene for antibiotic resistance, so they survive and replicate
  • This results in the production of millions of copies of the recombinant DNA, which may be isolated for use or transcribed by the DNA to produce a useful product
298
Q

What is a plasmid?

A

A small circular DNA element that can replicate independently of the chromosome. It is typically found in bacteria.

299
Q

What is the competency of a cell (in molecular cloning)?

A

How willing the cell is to take up a recombinant plasmid.

300
Q

What things can be done to increase a cell’s competency in molecular cloning?

A
  • Placing the bacterial cells in cold calcium chloride, then exposing then to a brief heat shock at around 42°C
  • Electroporation -> Exposing cells to an electric field, which temporarily forms holes in the cell membrane
301
Q

What are some important feature of plasmids that are chosen for molecular cloning?

A
  • Antibiotic resistance gene (Ampr) -> For selection of cells that have actually taken up the plasmid
  • Origin of Replication (ori) -> For replication
  • Multiple cloning site (MCS) -> Unique restriction enzyme sites necessary for insertion of DNA
  • Phage promoters (T7, SP6) -> Enable in vitro transcription of the recombinant DNA fragment
302
Q

What is PCR?

A
  • Polymerase chain reaction
  • The in vitro production of multiple copies of a selected DNA fragment, using a minimum of a very small amount of starting material
303
Q

Describe how PCR works.

A
  • DNA template is chosen
  • Oligonucleotide primers are designed and synthesised, so that they flank the target sequence
  • Reaction mixture contains DNA template, DNA polymerase, primers and free dNTPs
  • First, the reaction mixture is heated to about 95*C, which separates the double-stranded DNA, so that single-strand templates are exposed
  • Next, the reaction mixture is cooled to about 60*C, which allows the primers to anneal to their complementary sequences on the DNA -> This will allow the DNA polymerase to begin synthesis and will also prevent re-annealing of the strands
  • Then, the reaction mixture is heated to 72*C, which increases the activity of the DNA polymerase, allowing it to synthesis the complementary strand of DNA using the free dNTPs
  • This cycle of 3 steps can be repeated multiple times, to create several copies of the initial DNA template -> This happens exponentially until the reaction substrates are depleted
304
Q

What things are required for PCR?

A
  • DNA template
  • Oligonucleotide primers (flanking the target DNA)
  • DNA template
  • DNA polymerase
  • Primers
  • Free dNTPs
305
Q

What are the temperature changes in a PCR cycle?

A
  • 95*
  • 60*
  • 72*
306
Q

What is real time PCR? [EXTRA?]

A
307
Q

What is RT-PCR?

A

Reverse transcriptase PCR (do not confuse with real time PCR):

  • It is like normal PCR
  • But first a virus-derived reverse transcriptase is used to create a complementary copy of DNA (cDNA) from an RNA template, which is then amplified using PCR
  • It is often used alongside real time PCR to estimate the amount of a certain RNA present in a sample (real time PCR involves the incorporation of a fluorescent dye into the DNA after each cycle, giving a quantitative result)
308
Q

What mRNA molecules is RT-PCR very useful for and why?

A

Eukaryotic mRNA as they all share a common 3’end. A short poly (T) primer ‘TTTTTTTTTTTTTTTTT’ (oligo dT) can be used to reverse transcribe all mRNA into DNA.

309
Q

Describe how Sanger sequencing works.

A
  • Uses the single-stranded DNA template that is being sequenced, a DNA primer, a DNA polymerase, dNTPs and ddNTPs that are labelled fluorescently or radioactively
  • Mixture of the reactants is heated to separate the strands in the template DNA, then cooled to allow the primer to bind to a strand of the template
  • Upon heating, the polymerase synthesises the complementary strand (starting at the primer), using dNTPs.
  • The number of ddNTPs is relatively small compared to the dNTPs, so they are very infrequently added to the strand being synthesised. However, they lack the OH on the 3’ carbon, so they are unable to form phosphodiester bonds with the next dNTP or ddNTP. This means that when they are added to the strand, it can no longer be extended (chain termination).
  • This is repeated many times, and probability dictates that (with enough repeats), the chain should be terminated at every residue in the strand at least once.
  • All of the synthesised single strands are processed by electrophoresis, with a detector at the end that detects the labelled ddNTPs (e.g. a laser scanner that scans the fluorescent labels). The shorter strands pass through the gel faster and are detected by the detector sooner, meaning that the identity of the base at each position in the original DNA sequence can be determined.
310
Q

Describe why ddNTPs in Sanger sequencing cause termination of the chain.

A

They do not have an OH in the 3’ position, but have a H instead.

311
Q

What is the principle of NGS methods?

A
  • The DNA is fragmented into small pieces whic can be processed in parallel
  • The overlapping reads can be assembled to give the whole genome
  • So the method has a higher throughput.
312
Q

Give an example of a single molecule DNA sequencing platform.

A

Oxford nanopore

313
Q

What is sequencing depth in DNA sequencing? [IMPORTANT]

A
  • The number of unique reads that include a given nucleotide in the reconstructed sequence.
  • e.g. The base at position 27 may come up 20 times in DNA fragments in whole genome sequencing
314
Q

What is immunocytochemistry/immunohistochemistry?

A

A technique used to visualize the localization of a specific protein in cells by use of a specific primary antibody that binds to it.

315
Q

What is immunofluoresence?

A

Detection and localisation of a particular protein within a cell or tissue using a fluorescently-labelled antibody. It is essentially immunocytochemistry but with fluorescence.

316
Q

What does ELISA stand for?

A

Enzyme-linked immunosorbent assay

317
Q

Describe how ELISA works.

A
  • Antibody recognising a specific antigen is immobilised on surface of the wells
  • Sample containing the potential antigen is added. After incubation, the wells are washed, removing any non-captured material.
  • Antibody recognising the specific antigen and conjugated with enzyme is added. Detection solution is added and if enzyme is present solution changes colour.
318
Q

Draw how Western blotting works.

A
319
Q

What is immunoprecipitation?

A
  • A technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.
  • This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.
320
Q

What is alternative polyadenylation?

A
  • Many protein-coding genes have more than one polyadenylation site, so a gene can code for several mRNAs that differ in their 3′ end.
  • Since alternative polyadenylation changes the length of the 3′ untranslated region, it can change which binding sites for microRNAs the 3′ untranslated region contains.
321
Q

What is the signal hypothesis?

A
  • The signal hypothesis proposes that proteins destined for secretion, which involves the movement of the protein across a biological membrane, are originally manufactured with an initial sequence of amino acids that may or may not present in the mature protein.
  • It is to do with the movement of proteins.
322
Q

What are some examples of multiple-copy genes?

A

Genes for:

  • Histones
  • Ribosomal RNA

These are genes that are found multiple times in DNA.

323
Q
A

YEAR 1 - Biochemistry and Medical Genetics (Anton) (8 decks)

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