Polymerase Chain Reaction (PCR)

There are three stages to PCR: denaturation, heating, and extension.

In the first stage, denaturation, the reaction tube in the thermal cycler is heated to 95 degrees Celsius. The high heat denatures DNA by breaking the hydrogen bonds; the double helix is broken into single strands. In the second stage, annealing, the temperature is cooled to 60 degrees Celsius to allow DNA primers to form H-bonds with their complementary bases at 3' end at both strands. In the third stage, extension, the temperature is increased to 72 degrees celsius. Taq DNA polymerase binds to DNA template where primer has annealed. Complementary nucleotides are added to 3' end of both primers, thus catalysing synthesis of a new daughter strand. Chain reaction occurs as the products of previous reactions are used as the reactants in the next cycle.

Q: List the advantages and limitations of PCR

Advantages:
PCR is highly sensitive as it is capable of amplifying sequences from minute sequences of target DNA. It also has high fidelity as it produces the sequences required with a low error rate. It is also rapid as it can generate many copies within a short period of time. It is a quick and reliable method for detecting all mutations. Moreover, amplification can be done from unusual sources e.g. Egyptian mummies where the DNA quality is not good due to degradation.

Limitations:
Some knowledge of DNA/amino acid sequence of desired gene/protein is needed for the synthesis of flanking primers.
Non-target DNA sequence may be amplified instead, as primers are short and there is the possibility of many sequences complementary to primers. Also, Taq polymerase does not perform proofreading so there is the possibility of errors

Human Genome Project

Benefits:
It provides an extremely detailed map of the entire human genome which elucidates the genetic basis of diseases, hence allowing for the early detection of genetic diseases and improved diagnosis. The project enables novel molecular treatment such as gene therapy to be devised to cure genetic diseases.
Ethical Concerns:
It may detect genetic diseases early thus causing those affected to feel like social outcasts.
Employers and insurance agents may abuse the information, hiring workers based on genetic predisposition.

Q: Explain the functions of primers in PCR

Primers mark out a sequence of DNA to amplify. This sequence would be complementary to the target DNA strand which it attaches itself to. They also prevent the single stranded DNA from re-annealling by sticking to a part of the target DNA. They also allow Taq polymerase to work from a 5' to 3' direction, since the enzyme can only work with a free 3' end.

Q: Explain the effects of too high a temperature on an enzyme

If the temperature is too high, an increase in kinetic energy of the molecules will cause excessive vibrations of the molecules. This will disrupt the 3 dimensional structure of the enzyme, causing it to become a random coil. The enzyme is therefore denatured. The active site of the enzyme will be "lost" as it is no longer complementary to the substrate in size, shape and charge, thus the enzyme becomes nonfunctional as the enzyme-substrate complex cannot be formed.

Glycolysis

Glycolysis the breakdown of a glucose molecule into two pyruvate molecules. It is an example of substrate level phosphorylation. It occurs in the cytoplasm, whether in aerobic or anaerobic conditions.

Glucose > Glucose-6-phosphate > Fructose-6-phosphate > Fructose 1,6 biphosphate > 2 x phosphoglyceraldehyde > 2 x 1,3-biphosphoglycerate > 2 x 3-phosphoglycerate > 2 x phosphoenolpyruvate > 2 x pyruvate

NB
Glucose > Glucose-6-phosphate: 1 x ATP is used
Fructose-6-phosphate > Fructose 1,6 biphosphate: 1 x ATP is used
These two steps are known as the energy investment phase.

1,3-biphosphoglycerate > 3-phosphoglycerate: payoff of 1 x ATP per molecule (total 2 ATP)
2 x phosphoenolpyruvate > 2 x pyruvate: payoff of 1 x ATP (total 2 ATP)
These two steps constitute part of the energy payoff phase.

Net gain: 2 ATP per molecule of glucose
Also note that NAD is reduced to form NADH when phosphoglyceraldehyde reacts to form 1,3-biphosphoglycerate.

Q: Explain how protein synthesis can be controlled at a transcriptional level in prokaryotes

In prokaryotes, genes that encode enzymes of a metabolic pathway are usually clustered together on the chromosome in a region called an operon. A typical operon consists of regulatory sequences, like promoter, operator, terminator, and structural genes. Lac operon is an inducible operon that can be under negative control by lac repressor or positive control by CAP. In the absence of lactose, repressor binds to operator and blocks RNA polymerase from binding to promoter, thus no / low level of transcription of structural genes. In the presence of lactose, lactose will bind to repressor and change its conformation and prevent repressor from binding to operator, thus RNA polymerase can bind to promoter and transcription occurs. When both glucose and lactose are present, bacteria can selectively catabolise glucose instead of lactose. In the absence of glucose, the concentration of cAMP is high and cAMP binds to CAP and activates CAP, thus CAP binds to CAP-binding site and enhances rate of transcription. In the presence of glucose, concentration of cAMP is low and CAP not activated, thus CAP does not bind to CAP-binding site and transcription of structural genes occur as a low / basal rate.