In Vivo Out Of Revison Guide

Question 1

Describe step 1 - how the DNA fragment is inserted in a vector

Answer 1

1. Vector is something that is used to transfer DNA into a cell
2. The vector DNA is cut open using the same restriction endonuclease that was used to isolate the dna fragment containing the target gene. So the sticky ends of the vector are complimentary to the sticky ends of the DNA fragment containing the gene
3. The vector dna and DNA fragment are mixed together with DNA Ligase. This joins the sticky ends of the DNA fragment to the DNA vector sticky ends. (Ligation)
4. This is then called recombinant DNA

Question 2

Describe step 2 - the vector transfer the DNA fragment into host cells (out of textbook)

Answer 2

Plasmids and bacterial cells are mixed together in a medium containing calcium ions. The calcium ions and changes in temp make the bacterial membrane permeable allowing. Plasmids to pass through the cell surface membrane into the cytoplasm

Question 3

Step 3 - how do we identity transformed cells

Answer 3

1. Marker genes can be inserted into before at the same time as the gene to be cloned
2. Host cells are grown on agar plates. Each cell divides divides and replicated its DNA, creating a colony of cloned cells. Transformed cells will produced colonies where all the cells contain the cloned gene and the marker gene
3. The marker gene can code for antibiotic resistance - host cells are grown containing the specific antibiotic, so only transformed cells that have the marker gene will survive and grow.

Question 4

How can fluorescent markers be used

Answer 4

Take up gene by a jellyfish and then that contain the gene will produce GFP and will fluoresce under a microscope

Question 5

How do we make sure the transformed host cells produced proteins coded for by DNA fragments

Answer 5

Need to make sure the vector contains specific promoter and terminator regions

Promoter - tell mRNA polymerase to start transcription

Terminator - tell it to stop

Without the right promoter region the DNA fragment won’t be transcribed by the host cell and a protein won’t be made

(Promoter and terminator regions may be present in the vector DNA or they may have to be added in along with the fragment)