Question

P O words Question 53 10 pts Outline the major steps involved in recombinant DNA technology required to clone a gene into a plasmid and confirm that the cloning was a success by utilizing bacteria. 12pt v Paragraph Β Ι Ο Α T²v ... p O words Question 54 10 pts List and describe four mechanisms used during eukaryotic regulation of gene expression that are NOT found in prokaryotes. MacBook Pro 200 000 000 F4 0 F7 DII F8 F3 F5 F6 F9 < $ % & * C

Answer 1

• Isolation of Genetic Material

The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.

Since DNA exists within the cell membrane along with other macromolecules such as RNA, polysaccharides, proteins, and lipids, it must be separated and purified which involves enzymes such as lysozymes, cellulase, chitinase, ribonuclease, proteases etc.

Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified DNA.

• Restriction Enzyme Digestion.

Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are called ‘restriction enzyme digestions’.

They involve the incubation of the purified DNA with the selected restriction enzyme, at conditions optimal for that specific enzyme.

• Amplification Using PCR.

Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence using the enzyme – DNA polymerase in vitro.

• PCR reactions are run on ‘thermal cyclers’ using the following components:

1. Template – DNA to be amplified
2. Primers – small, chemically synthesized oligonucleotides that are complementary to a region of the DNA.
3. Enzyme – DNA polymerase
4. Nucleotides – needed to extend the primers by the enzyme.

• The cut fragments of DNA can be amplified using PCR and then ligated with the cut vector.

• Ligation of DNA Molecules

The purified DNA and the vector of interest are cut with the same restriction enzyme.

• Insertion of Recombinant DNA Into Host.

In this step, the recombinant DNA is introduced into a recipient host cell mostly, a bacterial cell. This process is ‘Transformation’.

• Isolation of Recombinant Cells.

• The transformation process generates a mixed population of transformed and non-trans- formed host cells.
• The selection process involves filtering the transformed host cells only.