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MGC - Genes
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1
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What indicates a promoter region in a DNA sequence?
TATA box and -35 element
2
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Where is the TATA box located?
Position -10 (e.g. 10 bases upstream of the start of RNA sequence)
3
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What position does the RNA start being made from?
\+1
4
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What is the sequence in a TATA box?
TATAAT
5
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What is the sequence in the -35 element?
TTGACA
6
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Where are the STOP signal sequences contained?
Terminator region
7
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How do differentiated cells vary in terms of RNA production?
* Synthesise different proteins
* Transcribe only required genes
* Different patterns of gene expression
8
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What is the RNA ‘life cycle’ after transcription?
1. RNA processing
2. Nuclear export
3. Protein synthesis
4. RNA degradation
9
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What 3 things happen during RNA processing?
Capping, polyadenylation, splicing
10
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What is the purpose of RNA capping?
(Me)Gppp at 5’ end. Protection and label for beginning
11
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What is RNA polyadenylation?
Poly(A) tail → AAAAA… at 3’ end. Protection, increased stability and marker of RNA age
12
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What is the RNA cap?
(Me)Gppp at 5’ end (7-Methyl guanosine)
13
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Which enzyme is responsible for polyadenylation of RNA?
Poly(A) polymerase
14
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What is a non-coding region of DNA called?
Intron
15
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What is a coding region of DNA called?
Exon
16
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What happens during RNA splicing?
Introns are cut out and Exons joined together
17
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What causes RNA to be degraded?
Poly(A) tail is shortened until an enzyme can cut off the cap
18
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What degrades RNA?
Ribonucleases (RNases)
19
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What do start codons mark?
Start of an Open Reading Frame (ORF)
20
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What do all proteins begin with?
Methionine
21
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Where is the start codon normally located?
First AUG downstream of mRNA cap and the Shine-Dalgarno sequence (AGGAGG)
22
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Where is the STOP codon located on mRNA?
First stop IN FRAME signals end of ORF
23
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What is an Indel mutation?
Insertions and deletions of one or more nucleotides
24
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What is a nonsense mutation?
Base substitution leading to a STOP codon
25
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What is a missense mutation?
Base substitution leading to a different amino acid
26
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What can an Indel mutation lead to?
Frameshift mutation
27
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What human beta-globin genetic mutations lead to anaemia?
Haemoglobinopathies
28
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What is thalassaemia?
Imbalances in alpha and beta haemoglobin subunit ratios
29
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What causes sickle cell anaemia?
Haemoglobin protein variants
30
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What is the large subunit of the ribosome made up of?
49 proteins and 3 rRNAs
31
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What is the small subunit of the ribosome made up of?
33 proteins and 1 rRNA
32
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What end of tRNA attaches to the amino acid?
3’
33
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How are tRNAs specific to different amino acids?
Anticodon complementary to mRNA sequence
34
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How are amino acids attached to tRNA?
Ester bond
35
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Which enzyme attaches the amino acid to the tRNA?
Aminoacyl tRNA synthetases specific to each tRNA/amino acid combo
36
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How many tRNAs can bind to a ribosome at once?
3 (3 docking sites, E, P and A)
37
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Where does the aminoacyl-tRNA enter the ribosome?
A site
38
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Where does the tRNA leave the ribosome?
E site (for ‘empty’ tRNA)
39
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What tRNA docking site is the amino acid attached to the chain in?
P
40
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What enzyme synthesises the new peptide bond in the ribosome?
Peptidyl transferase
41
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What attaches to the STOP sequence of mRNA in the ribosome?
Release factor
42
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How is DNA isolated?
* Cell lysing (SDS)
* Removal of other cell components (purification)
* Precipitating DNA
43
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How can DNA be precipitated?
Lowering solvation (adding ethanol or salt)
44
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How do ethanol and/or salt lower solvation?
Remove the water molecules associated with the DNA
45
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What wavelength do bases absorb radiation at?
260nm (UV)
46
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How can the H bonds between bases be broken without disrupting the backbone?
Heating, raising pH, chaotropic agents
47
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What is a chaotropic agent?
Very strong H bonds (e.g. formamide)
48
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How can reannealing be prevented?
Ice bath
49
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What is the hyperchromic effect?
Double stranded DNA absorbs less light than single stranded
50
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What causes the hyperchromic effect?
Stacked bases in double stranded absorb less light
51
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What is TM?
Temperature at which 50% of DNA is denatured
52
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What affects TM?
GC bonds require more energy to denature than AT
53
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What can a TM measurement be used for?
Characterising GC content
54
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What dyes can be used to see DNA on a gel electrophoresis plate?
Ethidium bromide, SYBR green or gold
55
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How do dyes allow DNA to be seen on a gel electrophoresis plate?
Intercalates between the stacked bases
56
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What does oligo- mean?
Few
57
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What is a ‘hot’ copy?
Radioactive
58
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What can hot DNA copies be used for?
Autoradiography
59
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What can an autoradiograph of an agarose gel be used for?
Identification of very small amounts of DNA (e.g. forensics)
60
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What is the upper size limit of gel electrophoresis?
20,000 base pairs
61
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How is DNA cut into fragments for gel electrophoresis?
Restriction endonucleases
62
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Where do restriction endonucleases cut?
Palindromic recognition sites
63
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How can specific fragments (e.g. genes) be identified in a mixture>
* Probes complementary to the fragment (Southern blotting)
* Selective amplification of the fragment (PCR)
64
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What is the process of Southern blotting?
* Run sample on gel
* Denature DNA (alkali)
* Transfer to membrane
* Add probe
* Wash
* Expose to a film
* Develop film
65
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What are the basic steps of PCR (3)?
* Heat to denature 94C
* Cool to anneal primers 55C
* Polymerisation 72C
66
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What is needed when running gel electrophoresis of RNA due to it being single stranded?
Formaldehyde (denatures any tertiary structure)
67
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How is Northern blotting different to Southern?
Uses RNA
68
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What enzymes are needed to make DNA from RNA?
Reverse transcriptases from viruses
69
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What is formed when making DNA from RNA?
Complementary DNA (cDNA)
70
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What is the sense strand in DNA?
Top, copying strand
71
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What is the antisense strand in DNA?
Bottom, non-coding, template strand
72
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Which DNA strand ‘becomes’ the copy?
Sense strand. RNA is made based on the antisense strand so sense strand is the same as mRNA except for T/U swap.
73
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How many base pairs are there in a transcription bubble?
17
74
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Is transcription or DNA replication quicker?
DNA replication
75
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What is an ‘activated precursor’ needed for RNA polymerase to work?
Nucleoside triphosphates
76
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What is the error rate of RNA synthesis?
1 in 10^4 (no proof reading)
77
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How are sugars joined in DNA?
5’ 3’ phosphodiester linkage
78
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Which end of the DNA chain is the start?
5’
79
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What rule dictates A-T and G-C with them in a 1:1 ratio to each other?
Chargraff
80
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What is the percentage of G (and so C) in human DNA?
20
81
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What is the percentage of A (and so T) in human DNA?
30
82
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What ensures damage to a DNA strand does not lead to information loss?
Redundancy
83
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What increases the stability of the double helix?
* H bonds
* Hydrophobic effect (no water in centre)
* vdW forces
* Hydrophilic interactions b/n phosphate and water
84
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How many chromosomes in human DNA?
23
85
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How is the DNA sequence usually written?
Top strand only, 5’ to 3’
86
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How is uracil different to thymine?
Missing a methyl group
87
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Who demonstrated semi-conservative replication?
Meselson and Stahl
88
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What forms when DNA replication begins?
Bubble
89
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What are the end points of the DNA replication bubble called?
Replication fork
90
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What marks a DNA origin of replication in the amino acid sequence?
Enrichment of T and A (easier to break 2 H bonds)
91
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How is the process of DNA replication sped up?
Multiple replication origins
92
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Which DNA polymerase is the major replication enzyme?
DNA polymerase III
93
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Which end does DNA polymerase III build DNA in?
5’ → 3’ (nucleotides attached to 3’ end)
94
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What reaction is catalysed by DNA polymerase III?
Elimination of 2 phosphates by breaking covalent bond
95
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What is the problem with replicating the top and bottom DNA strand?
Polymerase only works 5’ → 3’. Bottom strand is opposite direction
96
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How is the leading (top) DNA strand replicated?
Continuously
97
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How is the lagging (bottom) DNA strand replicated?
Discontinuously
98
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How is the lagging strand replicated?
Okazaki fragments formed and joined by DNA ligase
99
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Why can’t polymerase III begin replication alone?
Cannot initiate new strand synthesis
100
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How is DNA replication started?
RNA primer formed (10 bases long)
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