Lecture 17: Genetic variation & disease

Question 1

What is an allele?

Answer 1

An allele is an alternative form of a genetic variant.

Must be located at the same place in the gene.

Question 2

What is a monogenic trait? How are such traits usually inherited?

Answer 2

• An inherited trait/characteristic that is controlled by a single gene.
• Usually follow simple dominant/recessive inheritance patterns.
• Environment may influence exact presentation of trait. eg. if have freckles gene, environment can determine how many

Question 3

Explain the difference between a loss of function and gain of function allele.

Answer 3

Gain of Function Alleles:
- A gain of function allele causes the protein to work too well or do something new
- Often show a dominant inheritance pattern. (as the other allele will struggle to compensate and hide the new increased function)

Loss of Function Alleles
* A loss of function allele causes the protein to no longer work.
* Often show a recessive inheritance pattern (as other allele can sometimes compensate for the loss of function)

Question 4

Define genotyping:

Answer 4

• the process of determining the specific genetic alleles in an organism’s DNA.
• Many different methods can be used to determine genotype eg. PCR-RFLP, fluorescence-based methods (including gene chips), genome sequencing.

Question 5

What are restriction enzymes?

Answer 5

• Restriction enzymes are proteins isolated from bacteria that cut DNA (bacterial defense mechanism)
• Each restriction enzyme recognises a particular sequence and cuts that sequence at a particular base.
• Recognised sequence is short (4 to 6 bases) & palindromic (reads the same forwards & backwards).
• Enzyme cuts the same place on both DNA strands.

Question 5

What is polymerase chain reaction, and how can this technique be used in genotyping?

Answer 5

Definition:
PCR is a technique used to amplify specific DNA sequences, creating millions of copies from a small initial sample.

Steps:
* Denaturation: DNA is heated to separate the strands.

• Annealing: Reaction is cooled so Short DNA primers bind to the specific target sequences on each strand.
• Elongation: heat increased slightly so DNA polymerase can extend the primers to form new DNA strands.

Application in Genotyping:
PCR can be used to amplify regions of DNA that contain genetic variants or polymorphisms. By analyzing these amplified regions, researchers can determine an individual’s genotype and identify specific genetic traits or mutations.

Question 6

How are restriction enzymes useful in genotyping?

Answer 6

• Restriction enzyme is chosen based on ability to cut one allele of the variant, but not the other allele, e.g. Restriction enzyme cuts “A” allele, but does not cut “B” allele.
• Restriction Fragment Length Polymorphism (gel electrophoresis)
• DNA fragments (after PCR and restriction digest) are separated by size on a DNA gel.
• Smaller fragments travel further through gel.
• Cut DNA has two fragments, uncut DNA has
  one fragment.

Genotyping:
By comparing the fragment patterns, researchers can determine the presence of specific alleles and identify genetic variations.

Question 7

What is a polygenic trait? How are such traits usually inherited?

Answer 7

• An inherited trait/characteristic that is controlled by multiple genes. eg. height
• Do not follow an obvious inheritance pattern, i.e.
  - Common in a family, but can’t be categorised into dominant/recessive, etc.
  - Often talked about in terms of likelihoods or risks.
• Also usually influenced by the environment.

Question 8

Diploid organisms have two of each chromosome, this is called… ?

Answer 8

A homologous pair

Question 9

What are the reaction components required for a PCR?

Answer 9

• DNA Sample (template strand): DNA extracted from organism’s cells.
• Primers: Small segment of DNA (~20 to 30 bp long)
  complementary to specific sequence surrounding DNA of
  interest.
• Polymerase: Heat-tolerant DNA polymerase enzyme.
• Nucleotides (dNTPs): DNA nucleotides to build new DNA strands with.

Question 10

Describe Fluorescence-based genotyping:

Answer 10

Use different “coloured” probes to detect alleles.

Process:
* PCR primers are designed to amplify DNA surrounding genetic variant of interest.

• Probes are designed to specifically anneal to one of the two alleles.
• Each probe has a different fluorescent dye attached.
• As polymerase amplifies the DNA the probe is broken down & releases the dye.
• The specialised PCR machine (called a LightCycler) detects the colour of the dye upon release.

This allows graphs to be made and clusters to be identified to determine the presence of different alleles.

Genotyping “chips” use fluorescence-based methods to
assess multiple genetic variants at once (50,000 -100,000 variants)

Question 11

Describe PKU (monogenetic disease), include inheritance, effect on function, symptoms and remedies:

Answer 11

Inheritance:
Autosomal recessive; requires two recessive alleles to not posses phenylalanine hydroxylase (enzyme).

Effect on Function:
- phenylalanine hydroxylase is the first enzyme in the (Phe) breakdown pathway that converts phenylalanine (Phe) to tyrosine (Tyr).

• Loss of phenylalanine hydroxylase activity leads to a buildup of Phe and toxic metabolites, causing brain damage.

Symptoms:
Brain damage - Intellectual disabilities, neurological issues.

Remedies:
Managed with a low-Phe diet, avoiding high-protein foods like meat, fish, and dairy.

Question 12

Describe Retinitis Pigmentosa (monogenetic disease), include inheritance, effect on function, symptoms

Answer 12

Retinitis Pigmentosa is a monogenetic disease characterized by dominant inheritance. Retinol is always bound to the rhodopsin receptor but the receptor is not always active (ionic lock) however, mutations in the rhodopsin receptor interupt the ionic lock, leading to its constant activation. This results in rod cell death and progressive vision loss.