molecular techniques

Question 1

what is the significance of PCR?

Answer 1

PCR is a technique that can amplify a specific region of DNA from a trace sample.

Question 2

what are the conditions that are required for PCR to be carried out?

Answer 2

1. amplification in PCR needs to be carried out in vitro
2. heat is used to separate the 2 DNA strands
3. at least part of the sequence of the DNA sample must be known

Question 3

what are the components of the PCR?

Answer 3

1. double stranded DNA template, which contains the nucleotide sequence of interest
2. PCR primers, which are two sets of short SS DNA primers, that must be specific to the nucleotide sequence of interest, as they flank the sequence of interest and are complementary to the 3‘ end of both template strands
3. free dNTPs, which must be present in excess as raw materials for the synthesis of new DNA strands
4. thermostable DNA polymerase, which is isolated from a thermophilic bacterium. the enzyme must be stable at high temperatures and must not be denatured by repeated heat treatments
5. PCR reaction buffer, which contains ingredients like buffering salts, detergents, and MgCl2, which is the cofactor required for the activity of DNA polymerase

Question 4

what are the stages of the PCR cycle?

Answer 4

1. denaturation of DNA template, where the reaction mixture is heated to 95 degrees celcius for 30s, as at this temperature, the hydrogen bonds holding the two strands of DNA template are broken and the DNA template is denatured.
2. annealing of primers, where the reaction mixture is cooled to 54 degrees celcius for 1 min in the presence of a large excess of the two sets of DNA primers. cooling allows primers to anneal to the complementary sequences at the 3‘ end of SS DNA template via hydrogen bonds
3. extension of primers, where the reaction mixture is heated to 72 degrees celcius for 2 min, which is close to the optimum temperature of Taq polymerase. the annealed primers prime DNA synthesis using the four dNTPs, and extend DNA in the 5‘ to 3‘ direction

Question 5

what are the features of PCR?

Answer 5

1. the PCR is a chain reaction, because newly synthesised DNA strands will serve as templates or DNA synthesis in subsequent cycles
2. the PCR is specific, and only the sequence of interest is amplifies as primers are specific and do not attach anywhere else
3. after 30 cycles, essentially all the DNA molecules are exact copies of the sequence of interest, where the number of DNA molecules has increased exponentially to more than 1 billion

Question 6

what are the advantages of PCR?

Answer 6

1. PCR is extremely sensitive, and can amplify sequences from minute amounts of DNA
2. PCR is rapid and can be easily automated, as a single PCR cycle takes less than 5 minutes
3. PCR can permit amplification of specific sequences from which the DNA is badly degraded, or embedded in a medium from which conventional DNA isolation is difficult. this makes PCR suitable for molecular anthropology and palenontology studies

Question 7

what are the limitations of PCR?

Answer 7

1. due to the extreme sensitvity of PCR, any contamination of the reaction by non-template nucleic acids present in the laboratory environment, could cause non target sequences to be amplified instead
2. DNA polymerases used in PCR often lack 3‘ to 5‘ exonuclease activity, having an error rate of 1 in 10000 bases which are wrongly incorporated
3. DNA polymerases efficiently amplify DNA products up to a few thousand base pairs, but PCRs for longer products are less efficient due to enzyme activity loss and inaccuracies
4. in order to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence, some prior sequence information is needed
5. PCR can only be applied to amplify nucleic acids, but not proteins

Question 8

what are the principles of DNA gel electrophoresis?

Answer 8

1. electrophoresis separates charged molecules, such as nucleic acids and proteins, based on their different rates of migration in an electric field.
2. as the phosphate groups of the sugar phosphate backbone in DNA are negatively charged, DNA moves towards the positive electrode
3. DNA molecules have a constant charge desnsity and separate in porous agarose and polyacrylamide gels primarily on the basis of size.
4. 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.

Question 9

what are the practical applications of DNA gel electrophoresis?

Answer 9

1. to separate DNA fragments according to size
2. to determine the approximate molecular weight of the separated DNA fragments
3. to isolate individual DNA fragments
4. to determine if a PCR is successful

Question 10

what are the steps of DNA gel electrophoresis?

Answer 10

1. agarose powder is mixed with a buffer solution which helps to maintain the DNA in a stable form during electrophoresis. a gel tray with a gel comb is placed at one end to create wells in the gel. the gel is poured into the gel tray and allowed to cool and solidify
2. the gel tray is placed into an electrophoresis chamber filled with enough electrophoresis buffer solution to cover the gel, allowing an electric current to flow throgh the gel from electrodes. the gel comb is then removed
3. the DNA sampels are mixed with a loading dye. bromophenol blue migrates slightly faster than DNA, while xylene cyanol migrates slightly slower than DNA. glycerol increase the density of the DNA sample. each DNA sample is loaded into an individual well, with one well reserved for a molecular weight marker to allow for size determination of the bands
4. a DC power supply is connected to the electrodes, and DNA is forced to move by application of DC from the cathode to the anode.
5. DNA molecules are stained with methylene blue or ethidum bromide, to allow for visualisation of the position of the DNA bands within the gel. viewing of the bands can also be accomplished by autoradiography if the DNA is radioactively labelled

Question 11

what are the principles of nucleic acid hybridisation?1. nu

Answer 11

1. when kept for a prolonger period at a lower temperature of 65 degrees celcius to permit hydrogen bonds between complementary base pairs to reestablish, the two SS DNA strands readily anneal to each other to reform a double helix
2. nucleic acid hybridisation can occur between any two SS nucleic acid chains provided thet have a complementary nucleotide sequence. the greater the complementarity between the two SS molecules, the more favourable the hybridisation reaction
3. probes are SS DNA or DNA thay are radioactively, or chemical labelled to facilitate detection, and can be cloned from genomic or cDNA moelcules, PCR generated DNA fragments or chemically synthesised nucleic acid molecules

Question 12

what are the advantages of nucleic acid hybridisation?

Answer 12

1. complementary sequences present at a concentration as low as 1 molecule per cell can be detected
2. a probe hybridised only to nucleic acid molecules, carrying all of part of the complementary sequence
3. nucleic acid sequences do not need to be perfectly complementary to be able to hybridise, and the stringency of hybridisation can be controlled by varying the incubation temperature

Question 13

what are the practical applications of nucleic acid hybridisation?

Answer 13

1. to detect, characterise, and quantify specific base sequences of 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 and changes in gene expression profiles
4. to screen libraries of cloned DNA or bacterial clones to identfy clones carrying DNA insert of interest
5. to compare base sequences between two DNA samples

Question 14

what are steps in southern blotting?

Answer 14

1. agarose gel elecrophoresis
2. the gel is placed on a paper wick, which absorbs buffer solution from a resevoir. a sheet of nitrocellulose or nylon membrane which binds nucleic acids is laid over the gel, followed by a stack of blotting paper. Capilary action draws the buffer solution through the cel and nitrocellulose/nylon into the blotting paper. the alkaline condition of the buffer denatures the dsDNA into SS.
3. the nitrocellulose/nylon membrane is carefully peeled off the gel and incubated in a sealed bag containing radioactively labelled SS DNA or RNA probe, which has a sequence complementary to the DNA of interest. over a prolonger period under favourable conditions, the probe hybridises with the DNA of interest
4. the membrane is removed from the bag and washed to remove any unbound DNA probe. autoradiography is conducted, to show hybridised DNA which appears as bands