molecular techniques Flashcards
(78 cards)
1
Q
what does PCR stand for
A
polymerase chain reaction
2
Q
what is PCR?
A
PCR is an in vitro (cell-free method) technique that exponentially amplifies a target segment of DNA from a small amount of template DNA in a very short period of time.
3
Q
to conduct PCR, the reagents are mixed in
A
PCR tubes
4
Q
how small are the quantities used of reagents in PCR?
A
1-2 μL
5
Q
what apparatus is used to measure volumes of each reagent (PCR)
A
micropipettes
6
Q
what is a thermal cycle
A
an automated machine that heats and cools the to the precise temperatures. The PCR tubes are placed into it. The process of heating and cooling is programmed to cycle through a certain number of repeats determined by the scientist.
7
Q
list the reagents in PCR
A
- DNA template
- thermostable DNA polymerase (Taq polymerase OR Pfu polymerase)
- Deoxyribonucleotide Triphosphates (dNTPs)
- Buffer
- Forward (F) and Reverse (R) DNA primers
8
Q
DNA template purpose in PCR
A
contains the region of the DNA fragment to be amplified
9
Q
Taq polymerase purpose in PCR
A
it’s a thermostable DNA polymerase and therefore able to withstand the repeated heating and cooling during PCR. it can amplify a 1 kbp strand of DNA in roughly 30 seconds a 72°C.
10
Q
major drawback of Taq polymerase
A
One major drawback is its low replication fidelity. This is because it does not have a 3’ to 5’ exonuclease mechanism to replace a mismatch in the newly synthesized DNA strand (a.k.a no proofreading).
11
Q
Pfu polymerase purpose in PCR
A
This thermostable DNA polymerase proofreads DNA because it has 3’ to 5’ exonuclease mechanism to replace a mismatch in the newly synthesized DNA thus resulting in a lower error rate.
12
Q
is Taq polymerase a bacterium or enzyme
A
bacterium
13
Q
is Pfu polymerase a bacterium or enzyme
A
enzyme
14
Q
purpose of deoxyribonucleotide triphosphates (dNTPs) in PCR
A
dNTPs (dATP, dTTP, dCTP, dGTP) are used as the monomers for the synthesis of DNA.
15
Q
purpose of buffer in PCR
A
This solution provides a suitable chemical environment for reagents to function optimally e.g. optimum pH for DNA Polymerase (protein in nature). Usually MgCl2 (magnesium chloride) is added to act as a cofactor for DNA polymerase.
16
Q
purpose of Forward (F) and Reverse (R) DNA primers in PCR
A
These primers are short (usually 18 to 25 bp), single stranded DNA sequences that are complementary to DNA template sequences that flank the target DNA.
Primers provide 3’OH group for the DNA polymerase to elongate the target sequence.
17
Q
procedure of PCR in the thermal cycler (just name each step)
A
- denaturation
- annealing
- elongation/extension
- repeat
18
Q
describe denaturation in the procedure of PCR
A
Double-stranded DNA is denatured into single strand DNA by heating to 95°C for 1-2 minutes. The high heat breaks the hydrogen bonds that hold the two DNA strands together.
19
Q
describe annealing in the procedure of PCR
A
The temperature is lowered to 55°C for 1-2 minutes to allow annealing of primers to the single stranded DNA template. The two DNA primers anneal through hydrogen bonding to their specific sites flanking the target DNA region to be amplified.
20
Q
what can a wrong temperature during the annealing step result in
A
a wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random.
21
Q
what is the melting temperature (Tm) defined as?
A
The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in
the double-helical state and half are in the single-stranded states.
22
Q
typically, the annealing temperature is about how many degrees celsius below the Tm of the primers use?
A
3-5 degrees
23
Q
describe elongation/extension in the procedure of PCR
A
temperature is increased to 72°C for 2-3 minutes for synthesis of complementary strands. (Taq polymerase amplifies at a rate of 1000kp per minute)
Taq polymerase adds dNTPs, complementary to the template strand, to the 3’ –OH end of the annealed DNA primer, synthesizing the target sequence in 5’ to 3’ direction.
24
Q
describe “repeat” in the procedure of PCR
A
Steps 1-3 are repeated for 20-40 cycles.
25
the number of DNA molecules amplified can be calculated by
2ⁿ, where n is the number of cycles
26
why does the actual yield of a PCR product after a certain number of cycles eventually level off/plateau?
1. The amount of primers and dNTPs is finite and eventually activity decreases as primers and dNTPs are used up.
2. Even though the enzymes (Taq or Pfu polymerases) are thermostable, they do get denatured over time.
27
list the advantages of PCR
1. fast / rapid
2. easy to carry out
3. sensitivity
4. robustness
5. cell-free
6. accuracy in DNA replication
28
elaborate on how "fast / rapid" is an advantage of PCR
A PCR reaction that consists of 30 cycles generates 2³⁰ copies of the target DNA within 3 hrs in an automated thermal cycler. In contrast, cell-based DNA cloning take weeks to generate the same amount of target DNA.
29
elaborate on how "easy to carry out" is an advantage of PCR
Simply mix the reagents together and place in the thermal cycler machine, set the programmes and wait for 30 cycles to complete.
30
explain how "sensitivity" is an advantage of PCR
PCR is capable of amplifying sequences from minute amounts of template DNA.
31
explain how "robustness" is an advantage of PCR
A broad range of nucleic acid sources are suitable templates for PCR amplification. PCR can permit amplification of specific sequences from material in which the DNA is badly degraded or embedded in a medium from which conventional DNA isolation is problematic. Thus it is suitable for molecular anthropology and paleontology studies.
PCR has also been used successfully to amplify DNA from formalin-fixed or paraffin-embedded tissue samples, which has important applications in molecular pathology and, in some cases, genetic linkage studies.
32
explain how "cell-free" is an advantage of PCR
PCR is a cell-free method of DNA replication, and requires no cleanup of unwanted cellular debris or vector DNA (e.g. plasmid).
33
explain how "accuracy in DNA replication" is an advantage of PCR
the use of thermostable polymerase e.g. Pfu DNA polymerase, has improved PCR’s fidelity; giving error rates between 1 in 10,000 and 1 in 100,000.
In comparison, the accuracy of DNA replication in host cell is dependent on the type of host cell i.e. high mutation rate of certain bacteria.
34
list the limitations of PCR
1. size range of the DNA sequences
2. limitation to design of primers
3. non-specific priming
4. DNA polymerase errors
35
elaborate how "size range of the DNA sequences" is a limitation of PCR
PCR works readily with DNA of lengths 0.1 to 5 kb. Above this length the polymerase tends to fall off during the elongation process and the typical heating cycle is too short for polymerization to be completed. However, cell-based DNA cloning allows cloning up to 25 kb.
36
elaborate how "limitation to design of primers" is a limitation of PCR
To synthesize primers, the sequence flanking the DNA segment of interest must be known. Hence, PCR cannot be used to amplify an unknown sequence.
37
elaborate how "non-specific priming" is a limitation of PCR
The non-specific binding of primers is always a possibility due to sequence duplications and partial primer binding. This can result in the wrong sequence being amplified.
38
elaborate how "DNA polymerase errors" is a limitation of PCR
Taq polymerase lacks a 3' to 5' exonuclease activity and therefore lacks a proofreading mechanism which involves replacing any mismatched nucleotide in the newly synthesized DNA strand with the correct ones. This results in a high error rate thus altering large proportions of the final product. The problem may be overcome using the high fidelity Pfu polymerase.
39
similarities between PCR and DNA replication
- separation of double stranded DNA prior to synthesis of complementary strand
- require primer to provide the free -OH group on the 3' end to start elongation
- use of DNA polymerase to synthesise complementary DNA
- each DNA molecule will consist of an original strand and a newly synthesised strand
40
differences between PCR and DNA replication
- high temperature is used to break the hydrogen bonds between complementary bases of opposite strands in PCR instead of helicase
- DNA primer is used in PCR instead of RNA primer
- only the target sequence of DNA in between the two DNA primers are copied instead of the whole strand
41
what is gel electrophoresis?
Gel electrophoresis is a method that uses a gel as sieving medium to separates macromolecules (e.g. DNA, RNA and protein).
42
electrophoresis uses the principle of ___ to ___
Electrophoresis uses the principle of movement of charged particle through an electrical field in a gel to separate the macromolecules on the basis of size, shape and electric charge.
43
why is gel electrophoresis performed
for analytical purposes or used as a preparative technique to purify molecules prior for use in other processes e.g. PCR
44
result of gel electrophoresis can be further enhanced by what tests?
1. Southern blot (for DNA) * important
2. Western blot (for protein)
3. Northern blot (for RNA)
45
what is done before gel electrophoresis to turn a sample of DNA into DNA fragments?
adding restriction enzymes
46
how does a restriction enzyme work?
it recognises a particular sequence and cuts it
47
what is the place where the restriction enzyme cuts called?
a restriction site
48
the numbers and sizes of DNA fragments is determined by
the number of restriction sites the DNA molecule has
49
list the equipment and reagents needed for gel electrophoresis
1. an electrophoresis chamber and power supply
2. gel casting trays and sample combs
3. agarose gel
4. electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE)
5. Loading dye which contains glycerol and tracking dye
6. DNA Ladder
7. Ethidium bromide (EtBr)
8. Transilluminator
50
purpose of an electrophoresis chamber and power supply in gel electrophoresis
To provide electrical currents for the running of DNA samples.
51
purpose of gel casting trays and sample combs in gel electrophoresis
For the casting of gel, available in various sizes. Molten agarose is poured into the gel casting trays to form sample wells around the sample combs.
52
what is agarose gel
A type of polysaccharide that coils into helical bundles upon cooling, forming a gel with 3D mesh ideal for the separation of macromolecules.
53
purpose of agarose gel in gel electrophoresis
separation of macromolecules. gel percentage (0.5-2.0%) controls pore size and hence influences separation of macromolecules
54
purpose of electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE), in gel electrophoresis
Provide a medium with ions to conduct current from cathode (-) to anode (+).
55
purpose of loading dye which contains glycerol and tracking dye in gel electrophoresis
DNA, RNA and proteins are low in density and hence will not sink into the well. Glycerol is very dense, hence will help to hold the samples in the well, preventing them from floating up and out of the well.
Tracking dye allows visual monitoring of how far the electrophoresis has proceeded.
56
what is a DNA ladder + purpose of DNA ladder in gel electrophoresis
a solution of DNA molecules of different lengths used as a reference to estimate the size of unknown DNA molecules that were separated through the gel
57
what is ethidium bromide (EtBr) + purpose of ethidium bromide (EtBr) in gel electrophoresis
A fluorescent dye visible under UV light that intercalates between bases of nucleic acids and allows convenient detection of DNA and RNA fragments in gels.
58
what is a transilluminator + purpose of transilluminator in gel electrophoresis
An UV light box which is used to visualize ethidium bromide-stained DNA and RNA in gels.
59
procedure of gel electrophoresis
(good luck)
1. Prepare agarose gel (0.5 – 2.0%) cast and set up electrophoresis chamber.
(Sometimes, DNA samples are heated 2-3 min in 95°C water bath before loading, to separate the dsDNA into ssDNA. Samples are then placed on ice to prevent reannealing.)
2. Load the electrophoresis buffer to completely immerse the gel.
Load 2-3 µl of DNA ladder into the first well.
Prepare DNA samples by adding 3 µl loading dye to 20 µl sample or markers. Mix well.
Load 2-3 µl of each sample into the subsequent wells.
• The DNA is normally not visible during this process, so the tracking dye added to the DNA helps to estimate how far DNA has migrated in the gel (avoid having DNA samples from running entirely off the gel). The tracking dye has a low molecular weight, and migrates faster than the DNA. Therefore, as long as the marker has not run past the end of the gel, the DNA will still be within the gel.
3. Apply a current (100-120V). Let it run for 30-60 minutes.
• The DNA migrates toward the positive anode due to the negative charges on its phosphate backbone.
• Small / less dense DNA strands move faster, large / denser DNA strands move slowly through the agarose gel.
4. DNA in sample is now separated according to size and ready for visualisation using the transilluminator.
60
why is it that sometimes, DNA samples are heated 2-3min in 95°C water bath before loading?
to separate the dsDNA into ssDNA
61
how do you use tracking dye to estimate how far DNA has migrated in the gel?
The tracking dye has a low molecular weight, and migrates faster than the DNA. Therefore, as long as the marker has not run past the end of the gel, the DNA will still be within the gel.
62
what is the voltage of the current used in gel electrophoresis
100-120V
63
how long does gel electrophoresis run for?
30-60 minutes
64
which electrode does the DNA migrate towards? why?
positive, due to the negative charges on its phosphate backbone
65
which DNA strands move faster, and which move slower?
Small / less dense DNA strands move faster, large / denser DNA strands move slowly through the agarose gel.
66
how is DNA separated by gel electrophoresis?
DNA molecules are negatively charged due to the sugar phosphate backbone. Thus DNA molecules move from cathode (negative terminal) to anode (positive terminal). As the DNA molecules moves through the gel, they are separated based on size, shape and electric charge by the porous gel, which form a complex matrix of pores.
67
how is DNA separated by gel electrophoresis on the basis of size / length?
When the electric current is applied, longer DNA molecules move more slowly through the pores within the agarose gel while shorter DNA molecules move faster.
The shorter DNA molecules will form bands further from the loading wells whilst the longer DNA molecules will form bands nearer to the latter. Each band is a collection of DNA molecules of the same length.
68
how is DNA separated by gel electrophoresis on the basis of shape?
Depend on the shape of the DNA, i.e., linearised DNA, uncoiled plasmid and super-coiled plasmid, the speed and ease of movement of DNA molecules increases.
Linearised DNA will move the slowest since its length will prevent smooth passage though the gel. Uncoiled plasmid will move through the gel faster and more ease due to its shape of a circular DNA molecule. Super-coiled plasmid will move though the gel the fastest because of its compact structure. (Most of the time, this is not a factor of consideration for separation of nucleic acids since most samples studied will be linear.)
69
how is DNA separated by gel electrophoresis on the basis of electric charge?
In principle, more negatively charged molecules will move towards the anode faster than those less negatively charged. As all DNA and RNA has negatively charged sugar phosphate backbone, this is not a significant factor to consider for separation of nucleic acids. This is more significant for the separation of proteins.
70
why are some bands brighter than others in a gel electrophoregram?
more base pairs / DNA fragments
71
what is Southern blotting
This is a technique that transfers DNA molecules from agarose gel (after gel electrophoresis) to a nitrocellulose membrane to be labelled with a probe for further analysis.
72
what are some situations where Southern blotting is applied
• In certain circumstances, the amount of DNA on the gel is so much and/or so close together (overlapping each other) that it will look like a smear if visualized using ethidium bromide. Southern blotting is necessary to detect the presence of a specific sequences.
• The bands in different wells may be of the same length but different sequence. Southern blotting can be applied to distinguish the bands.
• It is useful for the analysis of repetitive sequences using a single probe.
• It is relatively quantitative and can be used to determine the number of copies of a gene in a genome.
• It can be used to study normal gene rearrangements in T-cell receptors or B-cell receptors / Immunoglobulin (Ig) gene.
73
describe step 1 of Southern blotting
The gel from the DNA electrophoresis is submerged in alkaline solution (typically containing sodium hydroxide) for 30min. Then wash the gel with distilled water.
• This is to denature the double-stranded DNA, separating them into single strands. Denaturation is necessary so that the DNA will stick to the membrane and be hybridized by the single stranded DNA probe.
74
describe step 2 of southern blotting
Place a sheet of nitrocellulose membrane on top of the gel and apply pressure evenly to the gel by placing a stack of paper towels and a weight on top. Let this assembly stand overnight.
• This causes the DNA to move from the gel onto the membrane by capillary action.
75
describe step 3 of Southern blotting
Bake the nitrocellulose membrane in an oven at 80 °C for 2 hours.
• This is to permanently crosslink the DNA to the membrane.
76
describe step 4 of Southern blotting
Submerge the membrane in a solution of labelled hybridization probe. Incubate overnight at 49°C.
• The probe is a short single stranded DNA molecule with a specific sequence that is complementary to the sequence of interest.
• The probe is labelled so that it can be detected. It is usually radioactively- or fluorescently-labelled. It may also be tagged with a chromogenic dye.
77
describe step 5 of Southern blotting
After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized.
• If radioactively-labelled probes are used, visualise on x-ray film by autoradiography.
• If fluorescently-labelled probes are used, visualise using fluorescent microscopy.
• if the probe is tagged with a chromogenic dye, develop the colour on the membrane.
78
describe the entire procedure of Southern blotting :D
1. The gel from the DNA electrophoresis is submerged in alkaline solution (typically containing sodium hydroxide) for 30min. Then wash the gel with distilled water.
• This is to denature the double-stranded DNA, separating them into single strands. Denaturation is necessary so that the DNA will stick to the membrane and be
hybridized by the single stranded DNA probe.
2. Place a sheet of nitrocellulose membrane on top of the gel and apply pressure evenly to the gel by placing a stack of paper towels and a weight on top. Let this assembly stand overnight.
• This causes the DNA to move from the gel onto the membrane by capillary action.
3. Bake the nitrocellulose membrane in an oven at 80 °C for 2 hours.
• This is to permanently crosslink the DNA to the membrane.
4. Submerge the membrane in a solution of labelled hybridization probe. Incubate overnight at 49°C.
• The probe is a short single stranded DNA molecule with a specific sequence that is complementary to the sequence of interest.
• The probe is labelled so that it can be detected. It is usually radioactively- or fluorescently-labelled. It may also be tagged with a chromogenic dye.
5. After hybridization, excess probe is washed from the membrane, and the pattern of
hybridization is visualized.
• If radioactively-labelled probes are used, visualise on x-ray film by autoradiography.
• If fluorescently-labelled probes are used, visualise using fluorescent microscopy.
• if the probe is tagged with a chromogenic dye, develop the colour on the membrane.
