Unit 3 AOS 1 Flashcards
(418 cards)
1
Q
define proteome
A
The complete set of proteins produced by a single cell or an organism
2
Q
define proteomics
A
technologies used in the study of the proteome, studies the protein patterns within a cell or organism
3
Q
what do proteomics allow for?
A
Proteomics allows us to study genes at work — by understanding proteins & how they interact in cells.
4
Q
what are the types of proteins?
A
- Structural
- Regulatory (Hormones & Receptors)
- Contractile
- Immunological
- Transport
- Catalytic (enzymes)
- Storage
5
Q
structural protein function?
A
- Forming the structural components of organs
- Support cell and tissue shape
6
Q
regulatory protein function?
A
Regulating cellular function (hormones)
- Many peptide hormones are chemical messengers used to communicate and induce changes in cells
- Receive signal from the environment
7
Q
contractile protein function?
A
- Forming the contractile elements in muscles
- Involved in the contraction and movement of muscles, the movement of internall cell contents around the cytoplasm, and the movement of cilia and flagella
8
Q
immunological protein function?
A
Functioning to combat invading microbes
- Involved in the immune system by recognising and destroying pathogens
9
Q
transport protein function?
A
- Acting as carrier molecules
- Typically embedded in membranes, controlling the entry and exit of substances from a cell
10
Q
catalytic protein function?
A
- Catalyzing metabolic reactions (enzymes)
- Enzymes are organic catalysts that speed up
chemical reactions
11
Q
storage protein function?
A
Act as reserves for metal ions and other molecules throughout organisms
12
Q
examples of structural proteins?
A
Collagen, keratin
- Keratin: a tough protein found in skin, hair, and nails
- Elastin: found in elastic connective tissues such as the skin
- Collagen: found in connective tissues such as tendons and ligaments
13
Q
examples of regulatory proteins?
A
Insulin, glucagon, adrenalin, human growth hormone, follicle stimulating hormone
- Insulin: regulates blood sugar levels
- Adrenaline: increases heart rate and expands airways
- Acetylcholine receptors
- Hormone receptors
14
Q
examples of contractile proteins?
A
Myosin, actin
- Myosin and actin: work together to enable muscle contraction
- Kinesin: moves along microtubules, enabling mitosis and vesicular transport
15
Q
examples of immunological proteins?
A
antibodies such as Gammaglobulin
- Antibodies (immunoglobulins)
- Complement proteins
16
Q
examples of transport proteins?
A
Hemoglobin, myoglobin
- Chloride channels
- Glucose channels
- Sodium-potassium pumps
17
Q
examples of catalytic proteins?
A
amylase, lipase, lactase, trypsin
- Catalase: breaks down hydrogen peroxide into water and oxygen
- Amylase: a digestive enzyme that breaks down starch into maltose
- RNA polymerase: catalyses the formation of mRNA from DNA
18
Q
examples of storage proteins?
A
- Ferritin: storage of iron
- Casein: storage of amino acids, carbohydrates, and minerals
19
Q
how do chemical reactions take place?
A
Chemical reactions in cells take place in a series of steps, called a biochemical pathway
20
Q
what do biochemical pathways do?
A
These complex pathways control and regulate cellular processes (reactions) that sustain life.
21
Q
features of biochemical pathways?
A
- Reactions convert substrate (reactants) into product.
- Are all ENERGY dependent
22
Q
what will happen to biochemical process at lower temperatures?
A
Will proceed more slowly at lower temperatures
23
Q
what will happen to biochemical process at higher temperatures?
A
Will proceed faster at higher temperatures
24
Q
what proteins are especially important for chemical reactions/biochemical pathways?
A
Enzymes are an important part of such processes.
25
what is a catabolic reaction?
Catabolism is the set of metabolic pathways that breaks down molecules into smaller units that are either oxidised to release energy or used in other anabolic reactions.
26
what is a anabolic reaction?
Anabolism is the set of metabolic pathways that construct molecules from smaller units, these reactions require energy.
27
what type of process is catabolism?
exergonic process
28
what type of process is anabolism?
endergonic process
29
what are enzymes?
Enzymes are proteins that help speed up metabolism, or the chemical reactions in our bodies.
30
what are the two things enzymes help to do?
They build some substances and break others down.
31
what organisms have enzymes?
- All living things have enzymes
32
are enzymes natural?
- Our bodies naturally produce enzymes.
- But enzymes are also in manufactured products and food.
33
what are enzymes also referred to as?
biological catalysts
34
What are enzymes structure?
Enzymes are mainly globular proteins, which have a generally rounded, ball shape due to their tertiary structure.
35
What is the active site of an enzyme?
The active site is a groove or pocket formed by the folding pattern of the protein, where substrate molecules bind and undergo a chemical reaction.
36
How do R groups of amino acids interact with the substrate?
R groups of amino acids lining the active site form molecular interactions with the substrate, weakening its bonds.
37
What effect do enzymes have on activation energy and how?
Enzymes lower the activation energy required for the reaction, as the R groups in the active site weaken the bonds.
38
What happens to the substrate during the reaction with enzymes?
The substrate breaks or is rearranged to form the products of the reaction.
39
What occurs after the products are formed in a chemical reaction with enzymes?
Products are released, allowing the enzyme to react again.
40
what are the two models of enzyme specificity?
- Lock and Key hypothesis
- Induced Fit Hypothesis (a hand in a glove model)
41
what is the Lock and Key hypothesis?
The active site is like a "lock" to which substrate fits like a "key".
The enzymes active site and substrate should fit like lock & key to initiate a reaction (E-S complex).
42
what is the Induced Fit Hypothesis?
Binding of substrate induces a conformational change in the active site & both adjust their shapes to provide optimal fit.
43
what model of enzyme specificity is most used today?
Induced Fit Hypothesis
44
What is the energy required for reactants to commence a reaction?
It is called the activation energy.
45
what is activation energy compared to?
This is like an energy barrier that has to be overcome before a reaction will occur.
46
What do catalysts do to activation energy?
They lower the activation energy.
47
What is the effect of lowering activation energy by catalysts?
It allows reactants to react faster to form products.
48
what do all reactants need to have before a chemical reactions occurs?
Energy
49
what do all catalyst do in relation to activation energy?
lower it
50
define enzymes
Enzymes are organic catalysts that lower the activation energy of reactions.
51
What are the characteristics of enzymes?
Enzymes are specific, not used up in reactions, can work in both directions, and can catalyze each step of metabolic pathways.
52
steps of enzymes in chemical reactions?
1. Substrate enters active site of enzyme
2. Enzyme changes shape slightly as substrate binds (Enzyme-substrate complex)
3. Reaction occurs
4. Products leaving active site of enzyme
53
How does temperature affect enzyme activity?
As temperature increases, so does the rate of enzyme activity until the optimum temperature is reached.
54
when is optimum energy reaches for enzymes in regard for temperature?
An optimum activity is reached at the enzyme's optimum temperature.
55
What happens to enzymes at high temperatures?
A continued increase in temperature results in a sharp decrease in activity as the enzyme's active site changes shape and becomes denatured.
56
What is denaturation in enzymes?
Denaturation is an irreversible change in the shape of the enzyme's active site.
57
What happens to enzymes at very low temperatures?
At very low temperatures, enzymes are inactive but can regain functionality when the temperature increases.
58
what is the optimum temperature for enzymes in humans?
37 degrees celsius
59
what does heat energy cause for enzymes and substrates?
Heat energy causes more collisions between enzyme and substrate
60
are enzymes sensitive to pH?
yes, very
61
How does pH affect enzyme activity?
Changing the pH of surroundings alters the shape of the active site, affecting enzyme folding and activity.
62
What happens to enzymes at low and high pH levels?
Enzymes become denatured at low and high pH levels.
63
What is the effect of substrate concentration on enzyme activity?
Enzymes work best with plenty of substrate; as substrate concentration increases, enzyme rate of activity increases.
64
What happens when substrate concentration continues to increase?
The same activity is reached when there are not enough enzyme molecules avaliable to break down excess substrate.
65
What is a limiting factor in enzyme reactions with plenty of substrate?
Enzyme concentration becomes a limiting factor when there is excess substrate.
66
What effect does increasing enzyme concentration have?
Increasing enzyme concentration speeds up the reaction as long as there is substrate available to bind to.
67
What occurs when all substrate is bound?
Once all substrate is bound, the reaction will no longer speed up as there is nothing for additional enzymes to bind to.
68
what are the types of enzyme inhibition?
- reversible enzyme inhibition
- irreversible enzyme inhibition
69
What is reversible enzyme inhibition?
Molecules that are reversible inhibitors bind to the enzyme non covalently and can dissociate from the enzyme.
70
What are the two forms of reversible inhibitors?
1. Competitive reversible inhibition
2. Non-competitive reversible inhibition
71
what is non-competitive reversible inhibition?
the inhibitor binds to part of the enzyme, away from the active site
72
what is competitive reversible inhibition?
the inhibitor competes directly with the substrate
73
How does non-competitive reversible inhibition affect enzyme activity?
It does not block the substrate, but inhibits catalytic activity by changing the conformational shape of the enzyme, decreasing the likelihood that the substrate will bind effectively.
74
What is irreversible enzyme inhibition?
Irreversible inhibitors bind covalently to the enzyme's active site, causing a permanent loss of catalytic activity. Toxic to cells.
75
What types of molecules act as irreversible inhibitors?
Heavy metal cations, nerve gases, and natural toxins.
76
how does competitive inhibition work?
In competitive inhibition, a molecule similar to the substrate competes with the substrate for the active site of the enzyme. The inhibitor is unable to be acted on by the enzyme.
77
What effect does competitive inhibition have on active sites?
Because of the presence of the inhibitor, fewer active sites are available to act on the substrate.
78
how does non-competitive inhibition work?
Non-competitive inhibition, also known as allosteric inhibition, occurs when an inhibitor binds to an enzyme at a site other than the active site. This site is referred to as an allosteric site.
79
What happens when an inhibitor binds to an enzyme in non-competitive inhibition?
The binding causes a conformational change in the active site of the enzyme, preventing the substrate from binding and the reaction from occurring.
80
What is a cofactor?
A molecule that assists enzyme functioning. Cofactors are essential for the activity of many enzymes.
81
what do some enzymes need to catalyse reactions?
Some enzymes require assistance from a cofactor to catalyse their reactions.
82
What are coenzymes?
A subset of cofactors that are organic, non-protein molecules.
83
role of coenzymes?
Coenzymes specifically assist enzymes in catalyzing reactions.
84
What happens to the structure of a coenzyme and enzyme when binding?
While the enzyme remains unchanged, the structure of the coenzyme is changed.
85
how do coenzymes help catalyse reactions?
Coenzymes bind to the active site and donate energy or molecules.
86
What occurs after a coenzyme participates in a reaction?
The coenzyme leaves the enzyme and is recycled by accepting more energy. This process allows the coenzyme to participate in multiple reactions.
87
What is referred to as the cycling of coenzymes?
The process where coenzymes are recycled after donating energy or molecules. Cycling of coenzymes is integral to certain biochemical processes.
88
True or False: Enzymes permanently change during the reactions they catalyze.
False. Enzymes remain unchanged after the reaction.
89
what are some common coenzymes?
- Adenine triphosphate
- Nicotine adenine dinucleotide (based on the vitamin niacin)
- Nicotine adenine dinucleotide phosphate (based on the vitamin niacin)
- Flavine adenine dinucleotide (based on the vitamin B12)
90
loaded form of Adenine triphosphate?
ATP
91
loaded form of Nicotine adenine dinucleotide?
NADH
92
loaded form of Nicotine adenine dinucleotide
phosphate?
NADPH
93
loaded form of Flavine adenine dinucleotide?
FADH2
94
unloaded form of Adenine triphosphate?
ADP
95
unloaded form of Nicotine adenine dinucleotide?
NAD+
96
unloaded form of Nicotine adenine dinucleotide
phosphate?
NADP
97
unloaded form of Flavine adenine dinucleotide?
FAD
98
function of Adenine triphosphate?
Energy transfer
99
function of Nicotine adenine dinucleotide?
Transfer of electrons and protons (only cellular respiration)
100
function of Nicotine adenine dinucleotide
phosphate?
Transfer of electrons and protons (only photosynthesis)
101
function of Flavine adenine dinucleotide?
Transfer of electrons and protons
102
what do proteins show?
Proteins show great functional diversity.
103
How can proteins be grouped?
They can be grouped into classes based upon their function within the cell.
104
are proteins important in relation to cells and organisms?
Proteins are key players in the chemistry of cells and the functions of organisms.
105
What roles do proteins play within cells?
Proteins serve as enzymes that control cellular processes and as fibres that provide structural support and movement.
106
What do proteins act as in multicellular organisms?
- as hormones to coordinate the functions of systems.
- to provide structural support as in bones and tendons
- as transport molecules for carrying oxygen,
- defense by being able to distinguish ‘self’ from ‘non-self’,
- and many other functions.
107
What is the significance of proteins in a cell?
Everything a cell is or does depends on the proteins it contains.
108
What are proteins made of?
Proteins are made up of linear polymers called polypeptides.
109
What are the constituent elements of proteins?
The constituent elements of proteins are carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sometimes sulfur (S) and phosphorus (P).
110
What are the subunits of proteins?
The subunits (monomers) of proteins are amino acids.
111
What is the 'R' group in amino acids?
The 'R' group is the part of amino acids that varies and determines the interaction between other molecules.
112
How many naturally occurring amino acids are there?
There are 20 naturally occurring amino acids, of which humans cannot synthesise 9.
113
What role does the 'R' group play in proteins?
The 'R' group plays an important role in establishing interactions between proteins.
114
what are the parts of an amino acid?
- amino group
- r group (side chain)
- hydrogen
- central carbon
- carboxyl group
115
What are proteins referred to as?
Proteins are referred to as polymers because they are made by chemically linking smaller units called monomers (amino acids).
116
How do amino acids join together?
Amino acids join together when a peptide bond forms between the amino group of one amino acid and the carboxyl group of the other.
117
What forms when amino acids join together?
When amino acids join in this way, a polypeptide forms.
118
What determines the order of amino acids in a protein?
The order of the amino acids is determined by the information coded in DNA.
119
what is a peptide bonds?
Peptide bond = CONH
120
what is a condensation reaction?
A dehydration synthesis catalyzed by a
polymerase enzyme to form a polymer, Amino acids join to form polypeptides by releasing H2O.
121
what is hydrolysis?
The splitting of a polymer by adding water to a covalent bond; catalyzed by a hydrolyase enzyme. Polypeptides can be broken down to their amino acids. This reaction requires H20 to be added.
122
What determines the 3D shape of a protein?
The characteristic 3D shape of a protein results from the coiling and folding of the polypeptide chain.
123
How does the shape of a protein affect its function?
The function of a protein depends upon the shape of the molecule. (Explains how enzymes work)
124
How many levels of organization does a protein molecule have?
A protein molecule has three or four levels of organization that determine its overall structure.
125
What is the primary structure of proteins?
The order or sequence of amino acids that has been assembled into a polypeptide chain on the ribosomes.
126
what is the linear primary structure of proteins determined by?
Linear sequence is determined by genetic material in nucleus.
127
What are the three folding patterns in the secondary structure of proteins?
The three folding patterns are α-helix, β-pleated sheets, and random coil.
128
what do the R groups of amino acids determine in secondary structure of proteins?
The R groups of the amino acids involved will determine the nature of the folding.
129
what happens to R groups as a result of secondary structure?
As a result of secondary structures, different R groups are brought closer together.
130
What is an α-helix?
An α-helix is a spiral molecule that is held together by hydrogen bonds between neighbouring CO & NH groups.
131
What are β-pleated sheets?
β-pleated sheets are folds in the polypeptide chain which are the result of hydrogen bonds between amino acids.
132
What is a random coil in protein structure?
A random coil is a shape that is neither α-helix nor β-pleated sheet.
133
What is the tertiary structure of proteins?
The tertiary structure involves the way that random coils, alpha helices, and beta sheets fold with respect to each other. The permanent functional shape or conformation.
134
what types of interactions occur in the tertiary structure and what do they determine?
Various types of interactions can occur between the R groups of amino acid residues. These interactions determine the tertiary structure of the polypeptide.
135
What defines the quaternary structure of proteins?
The quaternary structure describes complex patterns made up of more than one polypeptide chain, which may be the same or different types.
136
what bonds hold polypeptide strands together in quaternary structure?
Held together by hydrogen and disulphide bonds.
137
What are types of final products in quaternary protein structure and example?
- globular proteins such as haemoglobin
- fibrous proteins such as collagen
138
properties of enzymes?
- Catalytic Property
- Reversibility
- Specificity
- Heat Sensitivity
- pH Sensitivity
139
What is the nucleus?
The control centre of cells, enclosed by a double membrane.
140
What genetic material does the nucleus contain?
It contains genetic material called DNA.
141
What are nucleoli?
Large inclusions in the nucleus containing RNA, involved in the formation of ribosomes.
142
What does the outer membrane of the nucleus form?
The outer membrane forms the endoplasmic reticulum.
143
What are the two kinds of nucleic acids?
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
144
What are the constituent elements of nucleic acids?
Carbon (C), Hydrogen (H), Oxygen (O), Nitrogen (N), and Phosphorus (P).
145
What are the sub-units of nucleic acids?
Nucleotides made up of three parts: a sugar, a phosphate, and a nitrogen-containing base.
146
what does the base component of nucleotides comprise?
The base component of nucleotides which comprise the genetic code.
147
What forms the backbone of the DNA molecule?
The phosphate and sugar form the backbone, while the bases form the 'rungs'.
148
How many types of nitrogenous bases are there?
There are four types of nitrogenous bases.
149
what are the types of nitrogenous bases?
- adenine
- cytosine
- guanine
- thymine
150
what are the strands of DNA considered to each other?
anti-parallel
151
what bonds form between the nitrogenous bases?
hydrogen bonds
152
what is the structure of DNA called?
double helix
153
How do nucleotide polymers form?
By condensation polymerisation.
154
What bond is formed during the polymerization of nucleotides?
A phosphodiester bond is formed between the phosphate of one nucleotide and the sugar of another.
155
In which direction does the polynucleotide extend?
The polynucleotide extends in the direction 5’ → 3’.
156
what do cells use DNA and RNA for?
Cells use DNA to make RNA, and cells use RNA to make proteins.
157
what is RNA involved in?
This means RNA is involved in protein synthesis
158
how many types of RNA are there?
3 different forms exist each folded a different way
159
what are the types of RNA?
- Messenger RNA (mRNA)
- Ribosomal RNA (rRNA)
- Transfer RNA (tRNA)
160
what is mRNA's role?
Carries copied code of genetic information from nucleus to ribosomes in cytosol for protein synthesis
161
what is rRNA's role?
Helps make ribosomes and serves as the main structural component of ribosomes within cells.
162
what is tRNA's role?
Carry delivers specific amino acids after recognising specific nucleotide sequences (codons) on mRNA to ribosomes for protein formation
163
what happens to the structure of tRNA?
Hydrogen bonds create some areas of base pairing, causing folding in the strand
164
differences between DNA & RNA?
1. Different sugar:
- Deoxyribose (DNA)
- Ribose (RNA)
2. Different Nitrogen Bases:
- DNA: T (Thymine)
- RNA: U (Uracil)
3. Different Structure:
- DNA is double stranded
- RNA is single stranded
4. Storage:
- DNA is long term/inherited storage
- RNA is temporary molecules
165
similarities between DNA & RNA?
* nucleotides follow the same basic structure (phosphate group, five-carbon sugar, nitrogen-containing bases)
* contain the nucleotides adenine, guanine, and cytosine
* contain a sugar phosphate backbone
* follow the complementary base pairing rule: C pairs with G, A pairs with T (or U)
166
what is endocytosis and exocytosis?
Endocytosis and exocytosis are processes that deal with the exporting secretory macromolecules and engulfing material across the plasma membrane
167
what is a ribosome?
Site of protein synthesis
168
do ribosomes have a membrane?
Is not enclosed by a membrane
169
what are ribosomes made up of?
Made up of protein and rRNA
170
where are ribosomes found?
Can be found attached to endoplasmic reticulum (ER) or ‘free’ in the cytosol (in prokaryotic cells only free ribosomes exist)
171
what are the difference in proteins when synthesised at different places of ribosomes?
- When attached to ER (therefore Rough ER) the proteins produced are incorporated into membranes or are destined to be exported
- If ribosomes are ‘free’ the proteins produced are for local use
172
what is endoplasmic reticulum?
- Series of folded membranes and tubules found in the cytoplasm
- System of channels
173
what are the types of endoplasmic reticulum?
- Rough Endoplasmic Reticulum
- Smooth Endoplasmic Reticulum
174
what is Rough Endoplasmic Reticulum?
ribosomes attached to the external surface
175
what is Smooth Endoplasmic Reticulum?
no ribosomes
176
what happens to proteins in the endoplasmic reticulum?
Proteins produced by the ribosomes enter the Endoplasmic Reticulum tubules and are transported about the cell (then finally exported)
177
what is the golgi apparatus?
Storage depot for proteins that are to be secreted
178
what happens to proteins in the golgi apparatus?
- Proteins produced by ribosomes in the layers of Endoplasmic Reticulum are packaged into vesicles
- Vesicles are transported to Golgi complex and concentrated
- Proteins are then packaged into secretory vesicles & stored in cytosol before being released for exocytosis to occur
179
steps of the protein secretory pathways?
1. Ribosome - Synthesises proteins
2. Rough endoplasmic reticulum - Folds and transports proteins
3. Transport vesicle - Transports proteins
4. Golgi apparatus - Modifies and packages proteins
5. Secretory vesicle - Transports proteins
180
describe step 1 of protein secretary pathways?
The ribosomes are the sites of protein synthesis. They assemble polypeptide chains from amino acids by translating mRNA.
181
describe step 2 of protein secretary pathways?
If a protein is destined to be secreted, the ribosome synthesising it is usually attached to the rough endoplasmic reticulum rather than being free in the cytosol. The environment inside the rough endoplasmic reticulum allows for the correct folding of the newly formed polypeptide chain before being passed to the Golgi apparatus.
182
describe step 3 of protein secretary pathways?
A transport vesicle containing the protein buds off the rough endoplasmic reticulum and travels to the Golgi apparatus. The vesicle fuses with the Golgi membrane and releases the protein into its lumen.
183
describe step 4 of protein secretary pathways?
Proteins can have chemical groups (e.g. sugar molecules) added or removed at the Golgi apparatus, where they are often packaged into secretory vesicles for export or released directly into the cytosol for use by the cell.
184
describe step 5 of protein secretary pathways?
Secretory vesicles containing proteins for export bud off the Golgi apparatus and travel through the cytoplasm, fusing with the plasma membrane. This releases the proteins contained from within, into the extracellular environment through the process of exocytosis.
185
what is a gene?
A segment of DNA
186
what does a gene consist of?
A typical gene consists of tens of thousands of nucleotides
187
what are the roles of some genes?
- code for the production of a protein
- control the action of other genes
- determine our physical traits (phenotype)
188
what organisms is the genetic code found in?
Found in all organisms
189
what is the genetic code?
Genetic code the set of rules by which information is encoded in genetic material
190
what is coded information?
“Coded information” is the nucleotide sequences in the DNA template strands
191
what is decoded information?
“Decoded information” is the order of amino acids in polypeptides
192
what does the body use the genetic code for?
The body uses the genetic code to construct the correct amino acids and put them in the right sequence to create polypeptides
193
Properties of the genetic code?
- Universal
- Unambiguous
- Degenerate
- Non-overlapping
194
what is the universal property?
Nearly all living organisms use the same codons to code for specific amino acids.
195
what is the unambiguous property?
Each codon is only capable of coding for one specific amino acid.
196
what is the degenerate property?
- While each codon only codes for one amino acid (unambiguous), each amino acid may be coded for by multiple different codons (degenerate).
- This provides a degree of redundancy, where changes to the original DNA sequence through mutations may not necessarily lead to the insertion of a different amino acid.
197
what is the non-overlapping property?
Each triplet or codon is read independently, without overlapping from adjacent triplets or codons.
198
define Promoter
sequence of DNA to which RNA polymerase binds
199
define Leader region
the segment of DNA or mRNA that immediately precedes the coding region (involved in gene regulation)
200
define TATA box
a type of promoter region in eukaryotic cells (sequence of bases ‘TATAAA’)
201
define Introns
non-coding regions of DNA that do not code for proteins. They are spliced out during RNA processing
202
define Exons
regions of DNA that code for proteins and are not spliced out during RNA processing
203
define Termination sequence
a sequence of DNA that signals the end of transcription
204
what stages does eukaryotic gene expression involve?
- Transcription
- RNA processing
- Translation
- Post-translational modification
205
define Transcription
the process whereby a sequence of DNA is used as a template to produce a complementary sequence of mRNA
206
define Translation
the process where an mRNA sequence is read to produce a corresponding amino acid sequence to build a polypeptide
207
define Coding region
portion of DNA or RNA that codes for protein
208
define Messenger RNA (mRNA)
RNA molecules produced during transcription and carry genetic information from the nucleus to the ribosomes
209
define Start codon
sequence of 3 nucleotides in mRNA that signals the start of translation.
210
define Stop codon
sequence of 3 nucleotides in mRNA that signals the end of translation.
211
basics of transcription
RNA polymerase uses the DNA template strand to produce mRNA that is a transcript of the DNA coding strand.
212
what is important to remember about the bases in RNA?
Remember in RNA: U substitutes for T
213
what is the template strand?
gets transcribed and will be complementary to pre-RNA strand, 3' to 5'
214
what is the coding strand?
its sequence will be the same as that of the new RNA molecule, 5' to 3' (except U instead of T in RNA)
215
stages in transcription?
- Initiation
- Elongation
- Termination
216
what happens in the initiation stage of transcription?
with the help of transcription factors, RNA polymerase binds to the promoter region. RNA polymerase is then able to start transcription
217
what happens in the elongation stage of transcription?
RNA polymerase reads the template strand of DNA, to produce a complimentary single stranded RNA molecule known as pre-mRNA
218
what happens in the termination stage of transcription?
when RNA polymerase reaches a termination sequence it detaches and the pre-mRNA sequence is released
219
process of transcription according to VCAA?
* DNA unwinds/unzips
* RNA polymerase catalyses transcription through the joining of complementary RNA nucleotides
* transcription of the DNA template strand into pre-mRNA occurs
* pre-mRNA is complementary to the DNA template strand
* in the pre-mRNA, adenine (A) pairs with uracil (U), not with thymine (T).
220
what is RNA processing?
Pre-mRNA then undergoes post-transcriptional modification before it can leave the nucleus and move to the ribosome.
221
what occurs in RNA processing?
- the addition of a 5’ methyl-G cap and a 3’ poly-A tail
- the removal of introns and the splicing of exons together
222
final result of RNA processing?
- So the original pre-mRNA molecule is longer than the final mRNA molecule
- mRNA is now operational
223
what does capping help with?
Capping helps with mRNA recognition by the ribosome during translation.
224
what does the poly-A-tail help with?
The poly-A tail increases the stability of the molecule.
225
what does splicing help with?
Splicing makes genes more “modular”
226
what is alternative splicing?
Arranging exons in different patterns, called alternative splicing, enables cells to make different proteins from a single gene.
227
what happens to introns after they are removed?
Introns are usually degraded.
228
what and where do the two main steps of protein synthesis occur?
The two main steps in protein synthesis occur in separate compartments: transcription in the nucleus and translation in the cytoplasm.
229
where does mRNA move?
mRNA moves out of the nucleus, to the cytoplasm, through pores in the nuclear membrane
230
does protein synthesis occur in two different places in prokaryotic cells?
Prokaryotic cells have no nucleus, and the chromosome is in direct contact with the cytoplasm, and protein synthesis can begin even while the DNA is being transcribed.
231
basis of translation?
During translation, the mature mRNA molecule is decoded and translated into a sequence of amino acids, eventually forming a polypeptide chain.
232
where does translation occur?
Occurs in the cytoplasm on organelles called ribosomes
233
what are the stages of translation?
- initiation
- elongation
- termination
234
what does tRNA consist of?
Each ‘clover - leaf’ shaped tRNA molecule consists of a single strand of 76 nucleotides coiled and paired with themselves.
235
what are on the ends of the tRNA?
- At one end of each tRNA molecule are three bases that make up an anti-codon.
- At the other end of a tRNA molecule is a region which attaches to one specific amino acid
236
what happens in the initiation stage of translation ?
The 5’ end of the mRNA molecule binds to the ribosome and is read until the start codon (AUG) is recognised. This signifies the commencement of translation.
237
what is the start codon and what does it code for?
AUG and MET
238
what are the stop codons?
UAA, UAG and UGA
239
what happens in the elongation stage of translation?
The ribosome continues to read the mRNA bring the required tRNA (amino acids). Each binds to adjacent amino acids with a peptide bond via a condensation reaction
240
what happens in the termination stage of translation?
elongation continues until STOP codon is reached. The stop codon signals the end of translation as there are no corresponding tRNA molecules
241
process of translation according to VCAA?
* ribosome binds to and reads the mRNA molecule
* tRNA anticodons are complementary to the mRNA codons
* tRNA brings the corresponding amino acids to the ribosome
* adjacent amino acids are joined together into a polypeptide chain via a condensation reaction
242
key ideas of transcription?
* RNA polymerase binds to the
* DNA unwinds
* RNA polymerase reads the DNA template strand and uses complementary RNA nucleotides to catalyse the formation of pre-mRNA
* Transcription is terminated when the termination sequence is recognised
243
key ideas of RNA processing?
* Addition of a methyl-G cap to the 5' end
* Addition of a poly-A tail to the 3' end
* Introns removed and exons spliced together
244
key ideas of translation?
* mRNA molecule binds to the ribosome
* tRNA anticodons complementary to mRNA codons deliver corresponding amino acids to the ribosome
* Adjacent amino acids are joined with peptide bonds via a condensation reaction to form a polypeptide
* Translation ends when a STOP codon is recognised
245
location of Transcription?
Nucleus
246
location of RNA processing?
Nucleus
247
location of Translation?
Ribosomes
248
product of Transcription?
pre-mRNA from DNA template strand
249
product of RNA processing?
mRNA from pre-mRNA
250
product of Translation?
Polypeptide from mRNA
251
what is post translational modification?
Post translational modifications (PTMs) are processing events that change the properties of a protein
252
what do post translational modifications do?
PTM further modulate and extend the range of possible protein functions by attaching small chemical groups to an amino acid.
253
what is gene regulation?
Gene regulation involves the process of either inhibiting or activating gene expression.
254
why do we have gene regulation?
Organisms can prevent the unnecessary production of gene products (e.g. when they are not required), thereby conserving energy.
255
what are the two types of genes involved in gene regulation?
structural genes and regulatory genes
256
define structural genes
a segment of DNA that codes for proteins that play a role in the structure or function of a cell or organism
257
define regulatory genes
a segment of DNA responsible for producing proteins that control the expression of other genes
258
what is the link between structural and regulatory genes?
The important link between these two types of genes is that regulatory genes are responsible for controlling the expression of other genes, such as structural genes.
259
what is an operon?
An operon is a cluster of linked genes that all share a common promoter and operator and are transcribed at the same time
260
define operator
a short region of DNA that interacts with repressor proteins to alter the transcription of an operon. only in prokaryotes
261
define repressor protein
a protein coded for by a regulatory gene that prevents gene expression by binding to its operator
262
what is the trp operon?
The trp operon, found in E. coli bacteria, is a group of genes that control the production of the amino acid tryptophan, which bacteria need for their survival (albeit in very small amounts).
263
what does the trp operon consist of?
The trp operon is composed of a series of structural genes (trpE, trpD, trpC, trpB, and trpA). All 5 structural genes must be expressed for tryptophan to be produced.
264
when is the trp operon expressed?
The trp operon is expressed (turned "on") when tryptophan levels are low and repressed (turned "off") when they are high.
265
Differences in prokaryotic cells to eukaryotic cells in gene expression?
- Circular chromosomes (and no histones)
- No introns - no RNA processing needed
- mRNA has no methyl cap or poly A tail
- Have an operator region - between promoter and leader to help regulate gene expression. (Eukaryotes have no operator region)
- Transcription and translation occur simultaneously (not separately as in eukaryotes)
266
how do transcription and translation occur in prokaryotes?
Prokaryotic gene expression (both transcription and translation) occurs within the cytoplasm of a cell due to the lack of a defined nucleus; thus, the DNA is freely located within the cytoplasm.
267
how do transcription and translation occur in eukaryotes?
Eukaryotic gene expression occurs in both the nucleus (transcription) and cytoplasm (translation).
268
what are the two trp operon mechanisms?
- Trp operon repression
- Trp operon attenuation
269
what is Trp operon repression?
mechanism for gene regulation within the trp operon whereby repressor proteins stop the initiation of transcription when tryptophan levels are high
270
what does Trp operon repression respond to?
responds to the concentration of intracellular tryptophan
271
what is Trp operon attenuation?
mechanism for gene regulation within the trp operon whereby the premature ceasing of translation stops transcription when tryptophan levels are high
272
what does Trp operon attenuation respond to?
responds to the amount of tRNA-bound tryptophan
273
what happens when levels of intracellular of trp are high in the repression mechanism?
- When high levels of tryptophan (intracellular) are present, tryptophan binds to the repressor protein which induces a conformational change in the repressor protein.
- This allows the repressor protein to bind to the operator region.
- In this way, the repressor protein can prevent transcription of the structural genes by blocking the path of RNA polymerase, inhibiting unnecessary production of tryptophan.
274
what happens when levels of intracellular of trp are low in the repression mechanism?
- When tryptophan (intracellular) levels are low, there is an insufficient quantity of tryptophan molecules available to bind consistently to the repressor protein
- This causes the repressor protein to remain detached from the operator region, allowing RNA polymerase to transcribe the trp structural genes so that the level of tryptophan can increase.
275
what is the leader region in the trp operon?
The leader region of the Trp Operon has an attenuator region that is full of A & T (U).
276
what has the leader region got to do with base pairing?
Complementary base pairing between A & T is much weaker than the pairing of C & G.
277
what are the regions of the leader region?
There are 4 regions in the leader called Domains (1-4)
278
what is similar about the regions in the leader region?
Each domain has parts that are complementary to other parts so they can combine by base pairing to form hairpin loops
279
what is between domain 1 and 2 in the leader region?
Between Domains 1 & 2 there is a stop codon
280
what is in domain 1 in the leader region?
Within Domain 1 there are two consecutive codons for Trp
281
what domains can form hairpin loops?
- domain 1 and 2 can join together
- domain 3 and 4 can join together
- domain 2 and 3 can also join together
282
what does domain 1 contain?
domain 1 contains 14 codons that can be transcribed including two Trp codons and a stop codon
283
what is the attenuator comprised of?
an attenuator is found at the end of the leader region (made of UUUUUUUU that binds to AAAAAAAAA)
284
what does the attenuation mechanism do?
- Where the process of repression responds to the concentration of intracellular tryptophan, attenuation occurs in response to the amount of tRNA-bound tryptophan. This slows down transcription rather than stopping it as occurs in the repression mechanism.
- This is the mechanism where the leader region of the Trp Operon comes into play.
285
what happens when the cell has run out of free Trp (still has Trp bound to tRNA so no need to make more yet)?
- RNA polymerase runs along leader region of DNA & transcribes mRNA.
- At the same time a ribosome translates mRNA into proteins (while it is still being transcribed).
- Once ribosome reaches the stop codon, it stops there, but RNA polymerase continues to transcribe DNA until it reaches the attenuator region
- This causes domains 3 & 4 to form a hairpin loop (by complementary base pairing) and this creates tension that causes the mRNA molecule to separate at the attenuator region (which has weak bonds)
- RNA polymerase detaches from the DNA, causing transcription to stop before any structural genes are transcribed.
- Without all 5 structural genes, new tryptophan cannot be synthesised.
286
what happens when the cell has Trp completely absent (none on tRNA)?
- In this instance, when the ribosome gets to the two Trp codons (Domain 1), it stops as there is no Trp attached to tRNA.
- This means that ribosome stops at Domain 1
RNA polymerase continues to transcribe the DNA of Domains 2 & 3 and so these two regions overlap to form a hairpin loop.
- This looping allows RNA polymerase to continue transcription of the structural genes.
- This means the attenuator does not pull away and so it can complete the transcription of mRNA needed to produce all 5 structural genes needed to produce functional Trp.
287
summary of repression when trp is low?
* Regulatory gene is transcribed
* Repressor protein is unbound to operator
* Structural genes are transcribed
* Tryptophan is produced
288
summary of repression when trp is high?
* Regulatory gene is transcribed
* Repressor protein is bound to operator
* Structural genes are not transcribed
* No tryptophan is produced
289
summary of attenuation when trp is high?
* Regulatory gene is transcribed
* Ribosome does not pause, mRNA folds a different way
* Transcription stops and translation ends
* No tryptophan is produced
290
summary of attenuation when trp is low?
* Regulatory gene is transcribed
* Ribosome pauses, mRNA folds a specific way
* Transcription and translation continue
* Tryptophan is produced
291
tool for Synthesise DNA?
DNA Polymerase, DNA synthesisers and Reverse
Transcriptase (converts RNA to DNA)
292
tool for Cut DNA fragments?
Restriction enzymes
293
tool for Join DNA fragments?
Ligase
294
tool for Amplify DNA?
Polymerase Chain Reaction (PCR)
295
tool for Find particular DNA fragments?
Probes
296
tool for Separate DNA fragments by size?
Gel Electrophoresis
297
tool for Transform bacterial cells using recombinant DNA?
Recombinant plasmids
298
what are restrictions enzymes known as?
- Molecular scissors
- Known as endonucleases
299
where do restriction enzymes cut?
Cut DNA or RNA at specific recognition site
300
What is special about the specific restriction sites?
Base sequences at which endonucleases cut are palindromic, meaning the sequence of one strand is the same as the sequence of the complementary strand read backward.
301
how are endonucleases produced?
Produced naturally by bacteria as a defence against viruses, but they are enormously useful in genetic engineering for cutting DNA at precise places.
302
are endonucleases variable?
There are thousands of different restriction enzymes known, with over a hundred different recognition sequences.
303
how are restriction enzymes named?
Restriction enzymes are named after the bacteria species they came from
304
steps of endonuclease in bacteria?
1. A restriction enzyme cleaves the incoming phage DNA at recognition sites.
2. Other enzymes degrade the phage DNA into smaller fragments.
3. Methyl groups at the restriction sequence block the restriction enzyme and protect the bacterial DNA from being cleaved.
305
how do restriction enzymes cut?
They cut by breaking the bonds in a polynucleotide chain.
306
are restriction enzymes broad?
Each restriction enzyme cuts DNA at certain points, and is restricted to specific locations.
307
what is the site that restriction enzymes cut?
The positions where a restriction enzyme can cut is its recognition sequence/site and has a particular order of nucleotides.
308
what are the two types of cut?
- sticky ends
- blunt ends
309
what are sticky ends?
Most enzymes make a staggered cut in the two strands, forming sticky ends.
310
what are blunt ends?
Some restriction enzymes cut straight across both chains, forming blunt ends.
311
what segments do circular DNA make when cut?
2 recognition sites for a restriction endonuclease, 2 fragments of DNA, N=N
312
what segments to linear DNA make when cut?
2 recognition sites for a restriction endonuclease, 3 fragments, N = N + 1
313
what are ligases?
- Molecular glue
- Reverse role of endonucleases
314
what is the roles of ligases?
Ligases join or paste fragments of DNA or RNA together.
315
how do ligases join RNA/DNA together?
Ligases catalyse the formation of phosphodiester bonds between the two fragments (sugar-phosphate backbone) to merge them together
316
are ligases specific?
They are not specific and can join both blunt and sticky end pieces.
317
what can the joining of ends by ligases produce?
The joining can produce one longer piece of DNA or it can produce a circular molecule.
318
what are polymerases?
Polymerases synthesise polymer chains from monomers. Amplify (or multiply) sections of
DNA or RNA.
319
types of polymerases?
There are two types (RNA and DNA polymerases)
319
what is RNA polymerase used in?
RNA polymerase is used in transcription
319
what is DNA polymerase used in?
DNA polymerase is used in the replication of DNA or the amplification of DNA
319
what does DNA amplification require?
DNA amplification requires primers
320
what are primers?
short single stranded nucleic acid pieces that act as a starting point for polymerase to attach to
321
what direction does DNA/RNA synthesis occur?
Synthesis occurs in a 5’ to 3’ direction
322
what is CRISPR and what role does it play?
CRISPR is a naturally occurring sequence of DNA found in bacteria that plays an important role in their defence against viral attacks.
323
define Bacteriophage
a virus that infects prokaryotic organisms (e.g. bacteria)
324
what do bacteriophages do over time to a cell?
Over time, the virus replicates and causes the bacterium to lyse and die, spreading viral particles to infect other cells.
325
how did bacteria protect themselves against bacteriophages?
To protect themselves against bacteriophages, generations of bacteria slowly evolved the CRISPR-Cas9 system.
326
steps for bacteriophage infecting a cell?
- ATTACHMENT
- INJECTION
- SYNTHESIS & ASSEMBLY
- RELEASE
327
what does CRISPR stand for?
Stands for Clustered Regularly Interspaced Short Palindromic Repeats
328
what does Cas 9 stand for?
CRISPR associated protein 9
329
what is CRISPR-Cas9?
Is a unique and fairly new technology that enables scientists to edit genes.
330
how can scientists use CRISPR Cas-9?
Scientists can modify, add or delete specific nucleotides, delete sections of DNA or introduce a new gene altogether.
331
define CRISPR
gene editing system naturally found in bacteria that is now being used to edit genes in other organisms.
332
define Cas9
an enzyme that when attached to gRNA and can cut a target sequence of DNA
333
define gRNA
guided RNA which has a specific sequence determined by CRISPR to guide Cas9 to a specific site
334
define spacers
short sequences of DNA obtained from invading bacteriophages that are added into the CRISPR sequence
335
define PAM
(protospacer adjacent motif), sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9
336
what does the PAM sequence allow bacteria to ensure?
This sequence allows bacteria to ensure they can differentiate between self & non-self.
337
what did scientists find in bacteria in 1987?
In 1987 scientists studying bacteria noted that bacteria contained snippets of foreign DNA in their genome.
338
what does the CRISPR locus have?
- Upon closer inspection they noted that the CRISPR locus has short palindromic repeats that are identical in length and sequence.
- Within these repeats you have non repetitive spacer sequences.
339
what type of protein is Cas9?
Cas9 is an enzyme that can cut DNA (endonuclease).
340
what is different about Cas9 from other restriction enzymes?
While most restriction enzymes cut at a specific recognition sequence, the site at which Cas9 cuts is not fixed.
341
how does Cas9 work?
Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNA that is complementary to the guide RNA.
342
how does the gRNA work?
- The guide RNA (gRNA) is a molecule that is designed to find and bind to a specific sequence in the DNA.
- The gRNA is complementary to the target DNA sequence.
- Cas9 can then cut the DNA target sequence.
343
what is CRISPR in the Bacterial Genome?
short, clustered repeats of DNA found in prokaryotes which protect them against viral invasion
344
what do bacteria do upon infection?
Upon infection of a virus, bacteria collect small fragments of the viral DNA and insert these regions into their own genome (CRISPR loci).
345
what is throughout the CRISPR loci?
Throughout the CRISPR loci, viral sequences that have come from previous exposure to viruses are spaced with short repeats of host bacterial DNA.
346
what happens each time bacterium is exposed to a new virus?
Each time the bacterium is exposed to a new virus, a new piece of viral DNA is added to the 5’ end of the CRISPR locus.
347
what does transcription in CRISPR produce?
Transcription of the CRISPR locus produces RNA that are complementary to the integrated fragments of viral DNA.
348
what does gRNA from after it is transcribed?
This guided RNA forms a complex with the Cas9 proteins that can cleave and destroy the viral DNA.
349
what does the CRISPR system provide the bacterial cell with?
Much like receiving an immunisation, the CRISPR system provides the bacterial cell with a memory of previous viral infections that it uses to defend itself if reinfection were to occur.
350
what is the second criteria for Cas9 to cut other than matching gRNA?
That way anytime Cas9 searchers for a DNA sequence that matches its guide RNA, not only does it have to match but there must also be a NGG right next to it in the DNA.
350
process of CRISPR in bacteria?
1. First viral attack
2. Cas2 attacks viral DNA and exquisite a spacer
3. The new spacer is inserted into the CRISPR locus within the bacterial chromosome
4. Next viral attack
5. Upon next attack, gRNA is transcribed from the corresponding spacer
6. Cas9 was transcribed and translated to join gRNA.
7. The complex attack the viral DNA to destroy it
350
where do Cas1 and Cas2 cut out protospacers?
When Cas1 and Cas2 cut out protospacers from the viral DNA they always cut at a spot so that the adjacent sequence is NGG 5′-NGG-3′ PAM
351
how does Cas9 not cut itself?
The CRISPR array itself doesn’t contain NGG so its not at risk of being cut by Cas9.
352
is the PAM the same for all Cas endonucleases?
Please note that different Cas endonucleases can have different PAMs as can different species of bacteria.
353
CRISPR gene editing steps?
1. Synthetic gRNA is created in a lab that has a complementary spacer to the target DNA
2. A Cas9 enzyme is obtained with an appropriate target Protospacer Adjacent Motif or PAM (sequence of 2-6 nucleotides found next to the DNA targeted by Cas 9). The PAM site must contain the sequence NGG.
3. Cas9 and gRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex.
4. The gRNA-Cas9 mixture is then injected into a specific cell, such as a zygote.
5. The Cas9 finds the target PAM sequence and checks whether the gRNA aligns with the DNA.
6. Cas9 cuts the selected sequence of DNA.
7. The DNA has a blunt end cut that the cell will attempt to repair.
8. When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. Scientists may inject particular nucleotide sequences into the cell with the hope that it will ligate into the gap.
354
what does gRNA consist of and form?
Trans-activating CRISPR (tracr) RNA is a distinct RNA species that interacts with the CRISPR (cr) RNA to form the guided (g) RNA, and forms a gRNA-Cas9 complex.
355
what happens for DNA to be cut?
RNA-guided Cas9 nuclease recognizes the PAM sequence (5 -'NGG'-3) at the 3'-end of the target DNA region and creates DSBs at the 3 bp upstream of the PAM sequence.
356
what happens after DNA is cut by Cas9?
the DNA will naturally try and repair itself
357
what are the two mechanism for DNA repair in CRISPR?
- Non Homologous End Joining -NHEJ
- Homology directed repair
358
what is Non Homologous End Joining and what is the result?
break ends are directly ligated without need for a template strand, will result in errors forming (nucleotides could be deleted or added) resulting in the knockout or silencing of a gene.
359
what is Homology directed repair and what is the result?
HDR relies on a homologous repair template (often a sister chromatid) to repair the broken DNA, This process is often difficult and inefficient and scientists are still working to improve this process.
360
what does HDR allow scientists to do?
This allows scientists to edit a gene by introducing a piece of donor DNA that will assist the repair process.
361
Differences between CRISPR-Cas9 in bacteria and Gene Editing?
Purpose:
Natural - To attack and destroy invading viral DNA
Artificial - Induce mutations to alter genomic DNA
Production of gRNA:
Natural - Naturally through the transcription and post-transcriptional modifications of the CRISPR gene
Artificial - Synthetically produced in a laboratory
PAM sequence:
Natural - Specific to each host organism
Artificial - Cas9 enzyme can be altered to suit a specific gene
What happens after the cut:
Natural - DNA repair mechanisms often induce a mutation that inhibits viral function
Artificial - DNA can mutate to knock out, enhance, or otherwise change the function of genes
362
limitations of CRISPR?
- Fairly new technology, little success seen in human studies, more success seen in animal and plant studies.
- Substituting or adding nucleotides to a gene is not always easy or successful and unexpected mutations can occur.
363
ethical implications of CRISPR?
- Treating embryos is required before implantation
- Informed consent not obtained (when using embryos)
- Cost, only wealthy may access (who pays for this?)
- Discrimination (are certain traits seen as biologically inferior and who decides what needs ‘fixing’?)
364
Applications of CRISPR-Cas9?
- To cure a variety of genetic disorders e.g. sickle cell anaemia, thalassemia and cystic fibrosis.
- To help treat other diseases like cancer and AIDS.
- To ‘knock out’ genes to identify their functions.
- To snip out a faulty segment of a gene and replace it with a working copy.
- To activate or repress genes (e.g. activate cancer suppressor genes and silence cancer oncogenes).
- To add a new gene to the genome.
- To advance animal welfare e.g. hornless dairy cows.
- To modify crops and increase nutritional value.
- To create genetically modified food e.g. mushrooms that don’t brown and gluten free wheat.
- To locate certain genes by attaching fluorescent proteins to Cas9.
365
when is PCR used?
When only trace amounts of DNA are obtained, e.g. single hair root cell, a licked stamp, nose swab, dried blood stain, discarded cigarette butt, piece of chewing gum, drinking glass
366
what needs to happen when only small traces of DNA are found?
These trace amounts need to be amplified to millions of copies so that there is enough DNA for further investigation.
367
how is DNA amplified?
This is done by a process called Polymerase chain reaction (PCR).
368
how well can PCR amplify DNA?
PCR can amplify DNA quickly and accurately.
369
is PCR natural?
Is an artificial form of DNA replication
370
what materials does PCR use?
1. A DNA sample
2. A polymerase enzyme called Taq polymerase
3. Free Nucleotide bases (A, T, C, G)
4. DNA primers
5. Buffer solution
371
what is taq polymerase for in PCR?
Taq polymerases are enzymes found in bacteria that can withstand high heat temperatures and can be used to synthesise DNA or RNA.
372
what are DNA primers for in PCR?
these bind to the single stranded DNA and provide a starting point for DNA synthesis
373
what is the buffer solution for in PCR?
to provide a suitable chemical environment for Taq by maintaining the correct pH
374
facts about taq polymerase?
- Derived from bacteria Thermus aquaticus
- Heat stable DNA polymerase
- Ideal temperature is 72 degrees celsius
375
what are primers and what are they used for in PCR?
Single stranded short chains of nucleotides (up to 25 nucleotides long) that are complementary to the template strand. They join to the single strand of DNA allowing Taq polymerase to extend the DNA strand.
376
what are the two types of primers in PCR?
- Forward primer
- Reverse primer
377
what are forward primers?
Will bind to the start codon at the 3’ end of the template strand. This causes Taq polymerase to synthesise a new DNA strand in the same direction that RNA polymerase would function.
378
what are reverse primers?
Will bind to the stop codon at the 3’ end of the coding strand. This causes Taq polymerase to synthesise a new DNA strand in the reverse direction that RNA polymerase would function.
379
what are the PCR steps?
1. Denaturation
2. Annealing
3. Elongation
4. Repeat
380
what temperature does denaturation occur in PCR?
90 - 95°C
381
what happens during denaturation in PCR?
At this temperature the hydrogen bonds between the two strands of DNA are broken resulting in two separate strands of DNA.
382
what temperature does annealing occur in PCR?
50 - 55°C
383
what happens during annealing in PCR?
- The temperature is lowered to allow the primers to bind (anneal) to their complementary bases on each of the single strands of DNA.
- If temp is too low, double strands of DNA reform.
384
what temperature does elongation occur in PCR?
72°C
385
what happens during elongation (DNA synthesis) in PCR?
Taq DNA polymerase extends the DNA strand from the primers using the base pairing rule (in different directions – anti-parallel).
386
what happens in the repeat stage of PCR?
EXPONENTIAL AMPLIFICATION:
- The number of cycles is usually carried out 25 - 35 times but may vary upon the amount of DNA input and the desired yield of PCR product.
- If the DNA input is fewer than 10 copies, up to 40 cycles may be required to produce a sufficient yield.
387
Applications of PCR?
- DNA sequencing
- reverse transcription
- COVID-19 PCR test
388
how does PCR help in DNA sequencing?
- PCR with fluorescent, chain-terminating ddNTPs
- Size separation by capillary gel electrophoresis
- Laser excitation & detection by sequencing machine
389
how does PCR help in reverse transcription?
Reverse transcription (RT) is used to make complementary DNA (cDNA) from a mRNA template
390
how does PCR help in COVID-19 PCR tests?
1. Obtain Specimen: Swab.
2. Extract RNA from specimen and convert to DNA.
3. Amplify by PCR with SARS-CoV-2 specific primers.
4. Interpret results: presence of viral RNA indicates active SARS-CoV-2 infection.
391
when is gel electrophoresis used?
When DNA is cut with different restriction enzymes fragments of different sizes are formed.
392
what is gel electrophoresis?
Gel electrophoresis is one of the most useful means of separating DNA fragments according to their lengths for further analysis.
393
steps of gel electrophoresis?
- The DNA samples are placed into wells at one end of a thin slab of agarose gel.
- An electric current is passed through the gel.
- Each nucleotide in a DNA molecule contains a negatively-charged phosphate group, so DNA is attracted to the positive electrode.
- Hence wells are closest to the negative electrode
394
how does gel electrophoresis separate fragments?
- The molecules diffuse through the gel, and smaller lengths of DNA move faster than larger lengths.
- The smaller the length of a DNA molecule, the further down/along the gel it will move in a given time.
395
how can we see the movement in gel electrophoresis?
Dye is added to track the DNA as DNA fragments are invisible.
396
what happens after the current is turned off in gel electrophoresis?
- When the electric current is turned off a fluorescent dye (e.g. ethidium bromide) is then used to stain the DNA fragments so that the DNA becomes visible.
- Each band of DNA may contain many millions of fragments of the same size.
397
what is a standard ladder in gel electrophoresis?
A standard ladder contains a number of different DNA fragments with a known molecular size.
398
what is molecular size in gel electrophoresis?
Molecular size indicates the length of a nucleic acid sequence and is measured in base pairs (bp) or kilobases (kb).
399
how is DNA gel electrophoresis interpreted?
- Test DNA samples are then compared to the DNA of the standard ladder.
- DNA fragments that have migrated the same distance must be the same size.
- larger fragments travel less
- smaller fragments travel more
- thicker bands indicate a greater volume of fragments
400
what is a probe in gel electrophoresis?
A probe is a single-stranded DNA that is complementary to a specific sequence.
401
what is a probe used for in gel electrophoresis?
- A probe is added to help find a specific piece of DNA.
- The probe is added to the membrane and will join to the DNA band of interest.
- DNA-probe hybridisation occurs.
402
what is DNA profiling also called?
Also called DNA fingerprinting or DNA typing.
403
what is DNA profiling?
Forensic technique used to identify individuals by characteristics in their DNA.
404
applications of DNA profiling?
- Used in parental testing
- criminal investigations (e.g. to determine if people were present at a crime scene)
- immigration disputes
- identifying dead bodies
- finding lost relatives
- matching potential organ donors
405
how much of our DNA is similar?
Most of our DNA is similar (99% is the same).
406
does DNA profiling look at the whole genome?
DNA profiling does not involve sequencing the entire genome (costly and time consuming).
407
how is DNA profiling used?
DNA profiling is the process where a specific DNA pattern, called a profile, is obtained from a person or sample of bodily tissue. Even though we are all unique, most of our DNA is actually identical to other people's DNA. However, specific regions vary highly between people.
408
what happens in DNA profiling if only small amounts of DNA is available?
If only small amounts of DNA are available, PCR will need to be carried out first before a DNA profile can be produced.
409
DNA profiling process?
- A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR.
- DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments.
- Fragment length will differ between individuals due to the variable length of their short tandem repeats.
- The fragments are separated using gel electrophoresis and the resulting profiles are compared.
410
what is the most important thing for DNA profiling?
short tandem repeats
411
where ate STRs found?
Short tandem repeats (STRs) are found throughout the genome within genes and between genes, particularly near telomeres and centromeres.
412
what are STRs?
STRs consist of a variable number of tandem repeats of a 2 to 6 base pair sequence. In the example shown it is a four base sequence (AGAT) that is repeated.
413
how many STR sites in Australia are tested?
In Australia, there are 9 STR sites with each individual having a varied number of repeats, hence the unique profile of each person.
