Molecular Biology 1-3 Flashcards
(96 cards)
1
Q
What are nucleotides?
A
Monomeric units (building blocks) of DNA and RNA
2
Q
What are nucleotides comprised of?
A
A base, a sugar and a phosphate group
3
Q
What are the 2 types of nucleic acid?
A
Deoxybribonucleic acid (DNA) and ribonucleic acid (RNA)
4
Q
What are the functions of DNA and RNA?
A
- Direct synthesis of proteins
- Transmit genetic information
5
Q
What are bases?
A
Nitrogenous, aromatic, heterocyclic rings
6
Q
What are the 2 groups of bases?
A
Purines and pyrimidines
7
Q
Which bases are purines?
A
Adenine, guanine
8
Q
Which bases are pyrimidines?
A
Cytosine, thymine, uracil
9
Q
Which bases does DNA have?
A
Adenine, thymine, guanine, cytosine
10
Q
Which bases does RNA have?
A
Adenine, uracil, guanine, cytosine
11
Q
How many rings do purines have?
A
2
12
Q
How many rings do pyrimidines have?
A
1
13
Q
What type of sugar is in RNA?
A
Ribose
14
Q
What type of sugar is in DNA?
A
Deoxyribose (missing oxygen on C2)
15
Q
What is the difference between a nucleoside and a nucleotide?
A
Nucleosides don’t have a phosphate group - only base and sugar
16
Q
Describe the structure of a nucleoside
A
- Composed of a base and sugar (ribose or deoxyribose)
- Linked by N-glycosidic bond between 1’-C of sugar and N-9 of purine ring or N-1 of pyrimidine ring
17
Q
What are the bases in DNA?
A
Deoxyribonucleotides
18
Q
What are the bases in RNA?
A
Ribonucleotides
19
Q
Where is ATP produced?
A
In the mitochondria
20
Q
What is ATP?
A
Adenosine 5’ triphosphate
Main biological energy store
21
Q
What is cAMP?
A
Cyclic AMP
Adenosine -3’5’- monophosphate
A second messenger in the action of many hormones
22
Q
What is the sugar in ATP?
A
Ribose
ATP is a ribonucleotide
23
Q
What is the sugar in cAMP?
A
Ribose
cAMP is a ribonucleotide
24
Q
How is cAMP synthesised?
A
ATP is catalysed by adenyl cyclase, converted to cAMP and PPi and H+ ions (in the presence of Mg2+)
25
How is cAMP broken down?
cAMP and H2O catalysed to AMP and H+ by a phosphodiesterase (in presence of Mg2+ ions)
26
Describe how cAMP functions as a second messenger
1. Hormone binds to receptor in plasma membrane
2. Adenyl cyclase is stimulated
3. Increased adenyl cyclase activity leads to increased production of cAMP inside the cell
4. cAMP acts inside the cell to alter rate of one or more processes
27
What is the synthesis of cAMP catalysed by?
Adenyl cyclase
28
What is the breakdown of cAMP into AMP and H+ catalysed by?
A phosphodiesterase
29
Where is DNA found in the cell?
Mainly in the nucleus
30
Describe the structure of the DNA backbone
- DNA backbone consists of deoxyriboses linked by phosphodiester bridges
- Sugar of one nucleotide joined at C-3' (OH) to phosphate group attached to C-5' of sugar of the next nucleotide (3'-5' phosphodiester bond)
31
Describe the structure of DNA
- DNA is double stranded
- Structure held together by hydrogen bonding between base pairs
- 2 chains wound around each other to form a regular double helix
32
Which base does adenine pair with, and how many hydrogen bonds are formed?
Adenine with Thymine (or uracil in RNA)
| A 2 T (forms 2 hydrogen bonds)
33
Which base does guanine pair with and how many hydrogen bonds are formed?
Cytosine with Guanine
| C 3 G (forms 3 hydrogen bonds)
34
Where is RNA found in the cell?
Found primarily in the cytoplasm
35
Name 3 ways in which RNA differs from DNA
1. Uracil replaces thymine (pairs with adenine)
2. Ribose sugar replaces deoxyribose
3. Single stranded (no base pairing)
36
Explain what a 'hairpin loop' is in RNA, and how is this formed?
Local double stand formation can occur within RNA - RNA folds back on itself forming a hairpin loop. Local base pairing occurs.
37
When is the only time that RNA can have a small section of a double stranded loop?
When RNA folds back on itself forming a hairpin loop
38
What are the 3 types of RNA?
1. mRNA (messenger RNA)
2. tRNA (transfer RNA)
3. rRNA (ribosomal RNA)
39
What type of RNA makes up the majority of RNA in the cell?
rRNA - makes up 80% of RNA in the cell
40
What is the sedimentation coefficient (S)?
A measure of the relative sizes of RNA
41
What percentage of RNA is tRNA?
15%
42
What percentage of RNA is mRNA?
5%
43
Where is genetic information contained?
The base sequence in DNA contains genetic information (genetic code) in all cells
44
How is the genetic code transmitted?
The genetic code is transferred via genes
45
What are genes?
Specific stretches of DNA which code for a protein (average size = 1000-2000 bp)
46
What is the amino acid sequence determined by?
The nucleotide sequence of its corresponding gene
47
What is the whole genetic information within a single cell nucleus called?
Genome
48
What is the genome of a cell?
All the genetic information within a single cell nucleus
49
How many base pairs are in a human genome?
2,900,000 kilobases
50
Describe the stages of protein synthesis
1. In the nucleus, DNA unfolds (H bonds split) to expose base pairs on template stand
2. Transcription: RNA polymerase copies sequence of bases in template strand of DNA to produce mRNA
3. mRNA is translated by tRNA, using ribosome as a functional support (in cytoplasm)
51
What does tRNA do?
- Carries each amino acid to ribosome (one specific tRNA for each amino acid)
- Contains anticodon (3 bases) complementary to codon (3 bases) on mRNA
- Amino acid is transferred to growing polypeptide chain
- Sequence of mRNA codons determines the sequence of amino acids in protein
52
Describe the structure of a ribosome
Small and large subunits, contain proteins and rRNA
53
What happens to thymine when DNA is transcribe to mRNA?
Thymine replaced with uracil
54
How many amino acids are there?
20
55
How many tRNAs are encoded by triplet code?
4^3
| 64
56
The genetic code is degenerate. What does this mean?
A single amino acid may be coded for by more than one codon (each codon codes for a specific amino acid though)
57
How does a cell know when to start protein synthesis and when to terminate protein synthesis?
Start codon: AUG (codes for methionine)
The first amino acid in every protein is always Met
Stop codons: UAA, UAG, UGA
58
Which codon is the start codon?
AUG - codes for methionine
59
Which codons are stop codons?
UAA, UAG, UGA
60
What is a mutation?
Permanent alteration in a DNA (gene) sequence
61
What are the causes of a mutation?
- Errors in DNA synthesis that can occur spontaneously at low frequency
- Chemical mutagens
- Ionising radiation
62
What effects might substituting one base for another have?
- Conservative mutation: amino acid is replaced by one with similar properties
- Non-conservative mutation: amino acid is replaced by one with different properties
- No mutation: in many cases, a change in the third position of the codon does not change an amino acid
63
What is a conservative mutation?
Amino acid is replaced by one with similar properties
64
What is a non-conservative mutation?
Amino acid is replaced by one with different properties
65
What is the open reading frame (ORF)?
Part of the reading frame that has the ability to be translated
66
What happens during translation?
During translation, the cellular machinery reads each codon -> end up with a fully functional protein
67
What effect does insertion or deletion of a base have?
- Causes a frame-shift/open reading frame is shifted
- Whole sequence is changed - different type of protein will be translated
- If any of these changes introduces or removes a stop codon: premature or delayed termination
68
What type of mutation causes sickle cell anaemia?
Single base substitution: non-conservative mutation
GAG to GTG in the beta-chain of haemoglobin
Replaces glutamic acid with valine
69
What is a silent mutation?
A base mutation which does not alter the amino acid - in many cases, a change in the third position of the codon does not change the amino acid (genetic code is degenerate, amino acids are coded for by more than one codon)
70
What is gene cloning?
A process which allows the production of large numbers of copies of a particular piece of DNA
71
How are genes usually cloned?
- By isolating genes of interest using restriction enzymes, followed by gel electrophoresis and inserting them into plasmid
- Plasmid is then introduced into a bacterium and the bacterium grows producing large numbers of cells and hence many copies of the gene
- Gene can then be reisolated using the same restriction enzyme
72
What do restriction enzymes do?
- Cut double stranded DNA at specific DNA sequences
| - Sequences are typically 4-6 base pairs in length and "palindromic" i.e. they read the same in both directions
73
Define "palindromic"
Reading the same backwards as forwards
74
Most restriction enzymes make a "staggered cut". What does this allow?
Produces sequences with "sticky ends"
| Allows DNA fragments to re-associate by base pairing
75
What does DNA ligase do?
An enzyme that joins two strands of DNA together
76
What is gel electrophoresis?
A technique used to separate DNA fragments based on their size
77
Describe the stages in gel electrophoresis
1. Samples are applied to a gel immersed in buffer and a current is applied
2. Negatively-charged DNA migrates from the negative electrode (top of gel) to the positive electrode (bottom of gel)
3. Larger DNA fragments migrate more slowly than smaller fragments
78
Describe the procedure of gene cloning
1. A restriction enzyme is chosen that cuts on either side of the gene of interest
2. The gene is separated from other DNA fragments by gel electrophoresis
3. A suitable plasmid is linearised (cut at one point) using the same restriction enzyme
4. The cut plasmid and gene are mixed and the sticky ends of the plasmid and the gene are allowed to "anneal" (associate by base pairing)
5. The annealed ends are covalently joined using DNA ligase
6. The plasmid is reintroduced into bacterium
7. Bacteria are grown into a colony - antibiotic resistance genes in plasmid used to select colonies containing plasmids
8. Cloned cells are lysed and plasmids isolated by centrifugation
9. Plasmids cut with restriction enzyme releasing cloned gene
79
Why are plasmids containing gene of interest genetically engineered to contain antibiotic resistance genes?
To allow us to select only the bacteria that have taken up the plasmid of interest
80
What is the purpose of DNA sequencing?
Used to determine the base sequence of DNA
81
What are the 2 main types of DNA sequencing?
Sanger sequencing
| Next Generation sequencing
82
What is Sanger sequencing?
- A DNA sequencing method
- Used for small scale projects
- Synthesis of new strands complementary to a template strand in vitro
83
What is Sanger sequencing also known as?
The dideoxynucleotide chain termination method
84
What are the reaction components required for Sanger sequencing?
1. Single stranded DNA template
2. Primer
3. dNTPs
4. ddNTPs
5. DNA polymerase
6. Label
85
What is the single stranded DNA template in Sanger sequencing use for?
Its sequence is to be determined, used for the synthesis of a complementary strand
86
What is a "primer" in Sanger sequencing?
A short stretch of DNA (olignonucleotide ~20 bases long), serves as a primer for synthesis of the complementary DNA strand by elongation
87
What are dNTPs?
Deoxynucleotides: dATP, dCTP, dGTP, dTTP
| Building blocks of DNA
88
What are ddNTPs?
Dideoxynucleotides: ddATP, ddCTP, ddGTP, ddGTP
| Modified nucleotides that terminate DNA strand elongation
89
Which reaction component is required in Sanger sequencing to terminate the DNA strand?
Dideoxynucleotides: modified nucleotides
90
What does DNA polymerase do?
Catalyses DNA strand synthesis
91
Why is a label important in Sanger sequencing?
Required to visualise the products
92
What type of labels are used in Sanger sequencing to enable visualisation of the products?
```
Fluorescent labels (IRD800)
Radioactive (35S)
```
93
How do ddNTPs terminate DNA strand elongation?
- 3'-OH group of dNTPs replaced by an -H in corresponding ddNTPs
- Because they lack the 3'-OH required to form a phosphodiester bond with the next nucleotide, this results in chain termination
94
Why are ddNTPs added at a much lower concentration than dNTPs (ratio of 1:10 to 1:300)?
To allow strand elongation sufficient for sequence analysis
95
Outline the steps in DNA sequencing
1. DNA to be sequenced is mixed with primer
2. Primer binds to 3' end of DNA 3
3. DNA-primer mixture divided into 4 separate reaction tubes containing:
- all 4 dNTPS
- one of the 4 ddNTPs
- DNA polymerase
4. Chain synthesis then proceeds in each of the 4 reaction tubes
5. Gel electrophoresis for separation of reaction products
6. DNA bands detected by autoradiography or by laser in an automated sequencer
7. DNA sequence deduced from the pattern of bands in the 4 lanes
96
What is Next Generation Sequencing?
- High throughput sequencing
- No need for cloning, highly scalable
- Can sequence millions of genes and entire genomes at once
- Cheap and rapid
- Requires substantial bioinformatics analysis
