Lecture 14: Recombinant DNA Technology

Recombinant DNA Technology

• Recombinant DNA technology involves combining sequences that aren't normally found together (e.g., human and bacterial DNA).
• It's used to manipulate genes for expression and regulation studies, overexpress products, study protein structure and function, sequence genomes, cure diseases, and create transgenic animals.

Molecular Cloning

• Molecular cloning involves splicing DNA sequences from two different sources (a genome and a vector) to form a replicon containing the gene of interest.
• The process involves:
  • Isolating the gene of interest and generating fragments.
  • Inserting the gene into a vector (e.g., a plasmid) to create a composite molecule.
  • Introducing the composite molecule into living cells (e.g., bacteria) for propagation.
  • Selecting cells that contain the gene of interest.

Cloning

• A clone is a collection of cells that are all descendants of the same cell.
• Gene cloning involves producing many organisms carrying a gene of interest for study, expression, or insertion into other organisms.

Transgenic Plants and Animals

• Plants can be genetically altered to be resistant to glyphosate, insects, and to grow larger or have increased vitamins.
• Animals can produce compounds like medicines, vaccines, and metabolites.
• Examples:
  • Atryn, an anticoagulant, is produced in transgenic goats.
  • Spider silk is produced in transgenic goats.
  • Kanuma is used to treat lysosomal acid lipase deficiency and is produced in transgenic chicken eggs.

Gene Therapy

• Gene therapy involves using vectors to deliver therapeutic genes to cells.
• Achievements:
  • Cured lung cancer in mice.
  • Cured deafness in guinea pigs.
  • Cured sickle cell disease in mice.
  • Cured color blindness in monkeys.
  • Cured hemophilia in sheep using gene therapy and stem cell transplantation.
  • Cured Alzheimer's disease in mice using gene therapy.

Restriction Enzymes

• Restriction enzymes (endonucleases) cut nucleic acids at specific positions.
• They're used to analyze small genomes, distinguish or identify different DNA molecules, and verify insert directionality.

Restriction Enzyme Applications

• Restriction enzymes are used in:
  • Restriction mapping to determine the locations of restriction endonuclease cleavage sites.
  • RFLP (Restriction Fragment Length Polymorphism) to study inheritance, make pedigrees, detect disease markers, and in forensics.
  • Molecular cloning to open up the field of biotechnology.

DNA Polymerase

• DNA polymerase is essential for DNA replication and many other proteins.
• It can be used to copy DNA in PCR or fill in sticky ends.
• There are several types of DNA polymerase, with different activities, locations in the replication process, and proofreading capabilities.

Proofreading and Primer Removal

• DNA polymerases have several domains: polymerase, 3'→5' exonuclease (proofreading), and 5'→3' exonuclease (primer removal).
• Taq polymerase lacks a proofreading domain.
• The Klenow fragment is a DNA polymerase I fragment that lacks the 5'→3' exonuclease domain.
• T4 DNA polymerase is used to remove 3' overhangs and fill in 5' overhangs due to higher activity.### DNA Polymerases
• coli DNA pol I: 3'→5' exo, 5'→3' exo, unstable at high temperatures
• Klenow fragment: 3'→5' exo, unstable at high temperatures
• Exo- Klenow: No exonuclease activity
• Taq: Very fast (~100 bp/sec), but makes ~1 in 9000 mistakes, adds A overhangs
• Pfu: Slower (~10 bp/sec) but makes only 1 in a million mistakes, produces blunt ends
• Taq/Pfu: 16:1 ratio improves PCR, can amplify long targets
• Phusion high fidelity: High speed and accuracy
• Hot start: Polymerases that are inactivated by an aptamer or antibody

DNA Modification

• 5' and 3' Overhangs: Sticky ends can be converted to blunt ends by Klenow or T4 (higher activity)
• Klenow or T4: Removes 3' overhang, fills in 5' overhang

DNA Ligase

• Restriction fragments with compatible termini (overhangs or blunt ends) can be spliced together by DNA ligase
• DNA ligase: Catalyzes 3'-5' phosphodiester bond formation
• Bacteriophage T4 DNA ligase: Often used due to ease of purification, stability, and affordability
• Reaction conditions: Room temperature, requires ATP in the buffer

DNA Ligase Mechanism

• Ligase: Forms phosphodiester bond using ATP

Other Important Enzymes

• Topoisomerase: Relaxes supercoiled DNA, used in TOPO TA cloning
• Alkaline phosphatase: Removes 5' and 3' phosphates from DNA to prevent ligation
• T4 polynucleotide kinase: Phosphorylates DNA at 5' for ligation, probes, removes 3' phosphate
• Reverse transcriptase: Generates cDNA from mRNA