Describe a non-viral gene delivery system which can be used to deliver the functional [something] allele to stem cells.

1. Liposome-mediated gene transfer
2. Plasmid containing normal functional allele is inserted into the aqueous core of an artificial phospholipid bilayer sphere
3. The liposome fuses with the stem cell's plasma membrane, releasing the plasmid containing the normal allele into the cell's cytoplasm.

Why is [disease] suitable for treatment using gene therapy?

1. caused by a single gene
2. caused by recessive allele
3. ease of access to affected area
4. no invasive surgery needed

Describe how a specific gene mutation leads to cystic fibrosis.

A deletion of 3 base pairs occurs on the CFTR gene on chromosome 7, leading to a deletion of the codon coding for phenylalanine at position 508. The loss of phenylalanine disrupts the ATP binding site of the CFTR protein, which prevents binding of ATP and opening of the Cl^- channel, disrupting the diffusion of Cl^- out of the cell.

Explain the principles of RFLP.

1. Due to the polymorphic nature of DNA
2. genomic DNA from different individuals produce fragments of different lengths when digested by restriction enzymes,
3. resulting in a unique banding pattern among individuals

Characteristics of stem cells

1. Are unspecialised/undifferentiated
2. Able to differentiate
3. Can undergo extensive proliferation and self-renewal

Some definitions

What is recombinant DNA?
DNA that contains genes/sections of DNA from two species/two types of organisms.

What is a vector?
A carrier such as a virus or plasmid used to carry the gene of interest into another host/gene

Creating a cDNA library

1. Isolate mRNA from [tissue] 
2. Use reverse transcriptase to synthesise a cDNA strand using RNA as a template
3. Use DNA polymerase to make double-stranded cDNA and amplify using PCR
4. Cut plasmid with restriction enzyme that leaves blunt ends
5. Use terminal transferase to add extra cytosine nucleotides to the blunt end of DNA strands and add guanine nucleotides to that of plasmids to form sticky ends
6. Mix plasmid with cDNA; complementary sticky ends anneal to one another by hydrogen bonding;
7. Add DNA ligase to seal nicks by forming covalent phosphodiester bonds to form recombinant plasmid

What causes a unique banding pattern in Southern blots?

1. Polymorphic nature of DNA in individuals → variations in number/sequence of restriction sites/RFLPs
2. Resulting in unique banding pattern between individuals

Could also be for  gel electrophoresis.

How does Southern Blot work?

1. DNA fragments separated by size using gel electrophoresis
2. Gel slab containing DNA fragments laid under nitrocellulose membrane, set in alkaline medium to denature dsDNA into ssDNA. ssDNA is drawn upwards onto the nitrocellulose membrane and binds to the membrane.
3. Membrane incubated with probe complementary to the target sequence in the DNA fragments, which contains radioactive marker. Hybridization via complementary base pairing occurs.
4. The bands formed are then visualized using autoradiography. Radioactive regions expose the film, forming image that shows where the bands have formed complementary base pairs with the probe.

How gel electrophoresis works

1. DNA fragments are pipetted into the wells at the top of the gel furthest from the anode;
2. Dense loading buffer mixed with DNA sample to help it sink to the bottom of the well
3. Loading dyes added to allow visualization of separation process
4. Negatively charged DNA migrates out of well, toward anode when subjected to an electric field
5. Fragments migrate through an agarose gel matrix made out of a meshwork of polysaccharides, which impedes movement of longer fragments more than shorter fragments.
6. Longer fragments migrate slower compared to shorter fragments.