6C Genetic Change

Question 1

What is the process for creating recombinant DNA via invivo?

Answer 1

1. The required gene is isolated from a cell.
2. A plasmid is removed from bacteria.
3. The target gene and the plasmid DNA are cut using the same restriction enzyme.
4. The fragments produced have matching sticky ends (sections of single-stranded DNA with exposed nucleotide bases at the end)
5. The bacterial plasmid and target gene are added together.
6. As the sticky ends of the gene and the plasmid come together, they can join up via base pairing. This process is called annealing.
7. The DNA fragments are joined by the enzyme DNA ligase. Joined fragments can form a circular plasmid or a linear molecule.
8. The plasmid is inserted back into a bacterial cell, where multiple copies of the gene can be produced.

Question 2

Difference between blunt ends of a DNA fragment and sticky ends of DMA fragment?

Answer 2

Blunt ends -
Sections of DNA with no overhanging, exposed nucleotide bases. Restriction enzymes just chop straight down the middle. Blunt-ended fragments are harder to ligate together.

Sticky ends -
Sections of single-stranded DNA with exposed OVERHANGING nucleotide bases at the end. Two sticky end fragments can stick together by complementary base pairing. They hold two pieces of DNA together so they can be linked by DNA ligase.