molecular techniques

Question 1

What is the purpose of PCR (polymerase chain reaction)?

Answer 1

Amplify a specific region of DNA from a trace sample, meaning at least part of the sequence of the DNA sample must be known

Question 2

What are the components of PCR?

Answer 2

1. DNA template, containing the nucleotide sequence of interest
2. PCR primers: two sets of short, single-stranded DNA primers specific to the sequence of interest. they flank the sequence of interest and are complementary to the 3’ end of both template strands
3. Free deoxyribonucleoside triphosphates, present in excess as raw materials for the synthesis of new DNA strands
4. Thermostable DNA polymerase (Taq polymerase), which is stable at high temperatures and not denatured by repeated heat treatments. Optimum temp 75-80C
5. PCR reaction buffer, eg Mg2+ ions as cofactors required for DNA polymerase activity.

Question 3

Describe a single cycle of PCR.

Answer 3

1. Denaturation of DNA template (90-100C). Heating to 95C for 30s, breaks hydrogen bonds and DNA becomes single stranded
2. Annealing of primers (50-65C, not too high if not too much KE, primers cannot bind). Cooled to 54C for 1 min in presence of large excess of two sets of DNA primers. Allows primers to anneal specifically to complementary sequences at the 3’ end of ssDNA templates by hydrogen bonds — HYBRIDISATION
3. Extension of primers (60-75C, high temp for high rate of reaction). ~72C for 2 min (optimum temp for Taq polymerase). Prime DNA synthesis with the 4 deoxyribonucleotide triphosphates, 5’ to 3’.

Question 4

Describe how and why PCR has to undergo 20-30 cycles.n

Answer 4

Chain reaction because newly synthesised DNA strands serve as templates for DNA synthesis in subsequent cycles. The predominant DNA species is identical to the sequence of interest within a few cycles.
PCR is specific — only sequence of interest is amplified because primers are specific and do not attach elsewhere.
After 30 cycles, essentially all the DNA molecules are exact copies of the sequence of interest.

Question 5

What are the advantages of PCR?

Answer 5

1. Sensitivity: amplify sequences from minute amounts of DNA
2. Speed and ease of use: rapid and easily automated, overall takes 2-3 hours
3. Robustness: amplification from material in which DNA is badly degraded or embedded in a medium from which conventional DNA isolation is difficult. Can also amplify DNA from formalin fixed samples.

Question 6

What are the limitations of PCR?

Answer 6

1. Risk of contamination: due to extreme sensitivity, any contamination of the reaction by non-template nucleic acids could cause non-target sequences to be amplified instead.
2. Infidelity of DNA replication in vitro: Taq polymerase lacks 3’ to 5’ exonuclease activity (should not use long sequences to reduce chance of error)
3. Short size and limiting amounts of PCR product: PCRs for longer products are less efficient due to enzyme activity loss and accumulation of inaccuracies.
4. Need for target sequence information: In order to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence, some prior sequence information is needed.
5. can only be applied to amplify nucleic acids, but not proteins.

Question 7

Describe the principles of gel electrophoresis.

Answer 7

1. Electrophoresis separated charged molecules based on their different rates of movement in an electric field.
2. The electric field is created by application of a direct current through a semi-solid, porous gel matrix, made of a tangled meshwork of polymer chains of 2 main types: agarose - a polysaccharide extracted from seaweed, and polyacrylamide – made from monomers of a small organic molecule, acrylamide, that are polymerised and cross-linked.
3. The negatively charged phosphates groups of the sugar-phosphate backbone cause DNA molecules to move towards the positive electrode.
4. The complex network of pores in the gel matrix act as a “molecular sieve” to impede the movement of DNA molecules and separate them by size / length.
5. Shorter DNA fragments are less impeded by the pores than longer ones and move through the gel more quickly, so that DNA fragments of different lengths migrate as distinct bands. Thus, a complex mixture of linear DNA fragments is size-fractionated into discrete bands, each consisting of DNA fragments of the same length.
6. After electrophoresis, the gel is removed and stained with a DNA-binding dye such as methylene blue or ethidium bromide (EtBr, which intercalates DNA and fluoresces in UV light). This allows the separated DNA fragments to be visualised as a series of bands within the gel.

Question 8

Describe the practical applications of gel electrophoresis.

Answer 8

1. To separate DNA fragments (e.g. restriction digests) according to size.
2. To determine the approximate molecular weight of the separated DNA fragments.
3. To isolate / purify individual DNA fragments for further study (band(s) of interest can be excised from the gel).
4. To check results of PCR, i.e. to determine if a PCR experiment is successful

Question 9

Outline the steps of DNA gel electrophoresis.

Answer 9

1. Agarose powder mixed with buffer solution, maintains DNA in a stable form. Heated until agarose dissolves. A gel tray with a gel comb is placed at one end to create wells in the gel. The gel solution is poured into the gel tray and allowed to cool and solidify.
2. Set up is placed in electrophoresis chamber filled with enough buffer solution to cover the gel — allows electric current to flow. Gel comb is removed
3. DNA samples mixed with loading dye to make it visible. Bromophenol blue migrates slightly faster than DNA whereas xylene cyanol migrates slightly slower - monitoring of the rate of DNA movement / migration. Glycerol increases the density of the DNA sample so that they sink to the bottom of the wells.
   Each DNA samples is loaded into an individual well, near the negatively charged cathode. One well is reserved for a molecular weight marker that contains DNA fragments of known length, allowing for size determination of the bands
4. Application of electric field to start electrophoresis: DC power supply applied, forcing DNA molecules to move. Electrophoresis should be conducted long enough for the DNA molecules of different sizes to be well separated within the length of the gel. When the dye marker has moved a suitable distance (about 2/3 of the length of the gel) through the gel, the current is turned off and the gel removed from the tray.
5. Staining of gel to view separated DNA bands: stain gel with DNA-binding dye such as methylene blue or ethidum bromide, EtBr. Staining of the gel allows visualisation of the position of the DNA bands within the gel.

Question 10

Outline the principles of nucleic acid hybridisation.

Answer 10

1. Definition: Nucleic acid hybridisation is the process by which two complementary, single-stranded nucleic acid chains base-pair and reform a double-stranded hybrid.
2. DNA denaturation / melting: The DNA double helix can be separated into two single strands under conditions that disrupt the hydrogen bonds that hold complementary base-pairs together. (heating to ~100C, high pH of 13 or very low salt concentrations).
3. DNA renaturation / hybridisation: When kept for a prolonged period at a lower temperature of 65 °C to permit hydrogen bonds between complementary base-pairs to re-establish, the two single DNA strands readily anneal to each other to re-form a double helix.
4. Nucleic acid hybridisation can occur between any two single-stranded nucleic acid chains provided they have complementary nucleotide sequences. (centred on complementary base-pairing.)
5. Applications: detect specific DNA and RNA base / nucleotide sequences using specific single-stranded nucleic acid probes of known sequence.
6. Probe molecules:
    are single-stranded DNA or RNA ranging from 15 to several thousands of nucleotides long.
    are labelled i.e. carry radioactive, fluorescent or chemical markers to facilitate detection.
    can be cloned from genomic or cDNA molecules, DNA fragments generated by PCR or
   chemically synthesised nucleic acid molecules.
7. Advantages: Hybridisation reactions are highly sensitive (Complementary sequences present at a concentration as low as 1 molecule per cell) and selective (A probe hybridises only to nucleic acid molecules carrying all or part of the complementary sequence.)
8. Nucleic acid sequences do not need to be perfectly complementary to be able to hybridise. The stringency of hybridisation can be controlled by varying the incubation temperature for hybridisation.

Question 11

What are the practical applications of nucleic acid hybridisation?

Answer 11

1. To detect, characterise and quantify specific base / nucleotide sequences or genes in DNA / RNA
   molecules.
2. To locate particular genes of interest or families of related but non-identical genes in cells, tissues and organisms.
3. To study gene expression (e.g. whether a cell is transcribing a particular gene) and changes in gene expression profiles.
4. To screen (i) libraries of cloned DNA (library screening) or (ii) bacterial clones to identify clones / colonies carrying DNA insert of interest.
5. To compare base / nucleotide sequences between two DNA samples (e.g. from two different organisms or genomes) in phylogeny studies.

Question 12

Outline the principles of southern blotting.

Answer 12

The DNA within an experimental sample is separated on the basis of size by agarose gel
electrophoresis.
A replica of the DNA bands is made by transferring (“blotting”) the DNA in the gel onto a membrane
made of nitrocellulose or nylon.
The double-stranded DNA molecules must be denatured to their single-stranded form
before hybridisation with the probe; this is done either prior to or during the blotting process by exposing the gel to alkaline denaturing conditions.
The membrane is incubated in a solution containing the labelled, single-stranded DNA or RNA probe.
The DNA molecules that are complementary will specifically hybridise with the labelled probe. The DNA- probe hybrids are located by autoradiography or chemical / fluorescent means.
The size of the DNA that hybridise with the probe can be determined by reference to the molecular weight markers that were electrophoresed alongside the sample.

Question 13

Describe the steps of southern blotting.

Answer 13

1. Nucleic acids separated by size by GE.
2. The gel is placed on a paper wick, which absorbs the buffer solution (denatures the dsDNA) from a reservoir. A sheet of either nitrocellulose or nylon membrane which binds nucleic acids is laid over the gel, followed by a stack of blotting paper. Capillary action draws the buffer solution through the gel and nitrocellulose / nylon into the blotting paper.
3. The nitrocellulose / nylon membrane containing the bound DNA is carefully peeled off the gel and incubated in a sealed bag with a buffered salt solution containing radioactively labelled single-stranded DNA or RNA probe complementary to the DNA of interest.
   Over a prolonged period under favourable hybridisation conditions, the radiolabelled probe hybridises with the DNA of interest.
4.  The membrane is removed from the bag and washed thoroughly to remove any unbound DNA probe, so that only probe molecules that have hybridised to the DNA immobilised on the membrane remain attached.  Autoradiography: the DNA that has hybridised to the labelled probe shows up as bands on the autoradiograph. Only bands that contain a sequence complementary to the probe used (i.e. have undergone hybridisation with the probe) are visualised by autoradiography.