48. Recombinant DNA technologies (genetic engineering). Restrictases. Genomic cloning. DNA sequencing. Southern blot.

Question 1

Recombinant DNA technologies

Answer 1

• this technique allows taking out the sequence of interest and move it to another place
• form of artificial DNA made through the combination or insertion of one or more DNA strands
• insert is sequence of interest and vector is DNA molecule that accepts it
• Restriction enzymes are used to cut out the sequence of interest.

Manipulation:
1. Cleavage of DNA at specific sites by restriction nucleases, which greatly facilitates the isolation and manipulation of individual pieces of a genome.
2. DNA ligation
3. DNA cloning
4. Nucleic acid hybridization
5. DNA synthesis
6. Rapid determination of the sequence

Applications of recombinant DNA technologies in Medicine
- artificial insulin production
- drug delivery to target sites
- gene therapy- for prevention, treatment, or cure certain inherited disorders, such as: cystic fibrosis, alpha-1 antitrypsin deficiency, hemophilia, beta thalassemia, and sickle cell disease
- Antiviral therapy - detection the presence of HIV
- Vaccinations etc.

Question 2

Restriction enzymes (endonucleases)

Answer 2

• they cleave DNA at specific
  symmetrical nucleotide sequences
• both strands of DNA double helix are cut at specific points within target sequence
• Staggered cuts generate “sticky ends” that are short, single-stranded overhangs.
  They help cut DNA molecules rejoin through complementary base-pairing. This is crucial for DNA cloning
• restriction enzymes are usually obtained from bacteria, and their function is to protect the host genome against foreign DNA
• names reflect origin e.g. EcoRI comes from Escherichia coli

Question 3

Genome Cloning

Answer 3

• Genomic cloning involves cloning large DNA fragments to study DNA structure.
• Conducted in-vitro by isolating a specific DNA fragment from a donor organism.
• The isolated DNA fragment is introduced into a plasmid that replicates in a host cell.
• Plasmids are usually circular, double-stranded DNA found in certain bacteria, e.g., E. coli.
• Plasmids used in genetic engineering are called ‘vectors.’
• Phages are preferred vectors as they can incorporate large DNA fragments.
• Bacteriophages use bacteria as hosts to replicate, creating multiple copies of the DNA fragment.

Main steps involved in genomic cloning using Recombinant DNA Technology:
1. Isolation of DNA, i.e. free from other macromolecules.
2. Cutting gene atrecognition sites by restriction enzymes (restrictases)
3. Amplifying the gene copies through Polymerase chain reaction (PCR)
4. Ligation of DNA Molecules - joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase.
5. Insertion of Recombinant DNA Into a recipient host cell - this process is termed as Transformation.

Once the recombinant DNA is inserted into the host cell, it gets multiplied and is
expressed in the form of the manufactured protein under optimal conditions.

Question 4

DNA sequencing

Answer 4

• purified DNA fragment to be sequenced is distributed into 4 tubes and denatured.
• Primer is added, to bind to the vector DNA just prior to the insert.
• DNA polymerase and the 4 types of nucleotides are added
• dideoxynucleotide can be incorporated into DNA but is the last one