Unit 3. - Genetic Engineering Techniques Flashcards

Theoretical Foundations of Genetic Engineering

• Introduction to genetic engineering.
• Basic laboratory techniques.
• Enzymology for DNA manipulation in vitro.
• Molecular Cloning: Basic Vectors and Cellular Cloning Systems.
• Cloning Methods and Advanced vectors.
• Gene Libraries.
• Expression of cloned genes.
• Modification of DNA sequences: mutagenesis and gene editing.
• Synthetic Biology.

In Vitro DNA Modification

• Enzymes modify DNA in vitro, mimicking in vivo functions within a lab setting.

DNA Modifying Enzymes

• Nucleases: Degrade, shorten, or cut nucleic acid molecules.
• Polymerases: Copy DNA molecules.
• Other enzymes: Add or remove chemical groups.

Nucleases

• Degrade DNA by breaking phosphodiester linkages.
• Exonucleases: Remove nucleotides from the ends of a DNA chain.
  • Examples: Bal31 (Alteromonas espejiana), Exonuclease III (E. coli).
  • Bal31: Degrades both 3' and 5' termini of duplex DNA.
  • Lambda Exonuclease: Highly processive, acts in the 5' to 3' direction, removing 5' mononucleotides from duplex DNA; unable to initiate digestion at nicks or gaps.
• Endonucleases: Break internal phosphodiester bonds.
  • Examples: S1 nuclease (Aspergillus oryzae) for short ssDNA, DNase I (Bovine pancreas) for dsDNA and ssDNA, Restriction endonucleases.
• Other Nucleases:
  • RNAse A (Bovine pancreas): Degrades ssRNA, used in DNA extraction.
  • RNAse H: Endo- and exoribonuclease activity specific for RNA:DNA hybrids, used in RT-PCR to remove RNA after cDNA synthesis.

Polymerases

• Synthesize new DNA or RNA strands using a template.
  • Thermostable Polymerases (e.g., Taq, Pfu):
    • Catalyze 5'-3' polymerization of DNA from a primer.
    • Taq lacks 3'-5' exonuclease activity (no proofreading).
    • Pfu has 3'-5' exonuclease activity (proofreading).
    • Applications: PCR, sequencing.
  • DNA Polymerase I (E. coli):
    • In vivo: Eliminates RNA primer, fills gaps between Okazaki fragments.
    • Has 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease activities.
  • Klenow Fragment:
    • Lacks 5'-3' exonuclease activity, retains 3'-5' exonuclease activity (proofreading).
    • Applications: Filling nicks/holes in dsDNA, filling 5’ protruding dsDNA ends.
  • Reverse Transcriptase (RT):
    • 5'-3' DNA polymerase activity on RNA template.
  • RNA Polymerases (DNA-dependent):
    • Phage RNA polymerase (T7, T3, SP6): High specificity for phage gene promoters, used in in vitro RNA synthesis.
    • RNA polymerase from E. coli: Used in in vitro transcription with suitable promoters.
    • RNA polymerase II (wheat): Used in eukaryotic transcription studies.

Other DNA Modifying Enzymes

• Alkaline Phosphatase:
  • Removes phosphate group from 5' DNA end.
  • Applications: Prevents vector religation during cloning, dephosphorylation prior to radiolabeling.
• Polynucleotide Kinase:
  • Adds phosphate group to 5' end.
  • Applications: Radioactive labeling of probes, phosphorylation of oligonucleotides (primers).
• Terminal Transferase:
  • Adds dNTPs to 3'OH end of ssDNA or dsDNA.
  • Applications: Creation of homopolymer tails, radioactive labeling.

In Vitro DNA Cutting: Restriction Enzymes

• Bacterial protection against bacteriophages.
• Work with DNA methylases to protect bacterial DNA.

Type II Restriction Endonucleases

• Used in genetic engineering.
• DNA methylase domain separated from endonuclease domain.
• Nomenclature: genus, species, strain, and order of isolation (e.g., EcoRI).
• Recognition sites: 4-8 base pairs, symmetrical, often palindromic.

Restriction Enzyme Types

• Type II: Most work as homodimers; endonuclease and methylase activities are on different molecules.

Restriction Enzyme Nomenclature

• First letter: Genus (e.g., E for Escherichia).
• Second and third letters: Species (e.g., co for coli).
• Fourth letter: Strain (e.g., R).
• Roman numeral: Order of isolation (e.g., EcoRI, EcoRV).

Restriction Site Characteristics

• Recognition sites are typically 4-8 base pairs long.
• Sites are symmetrical; bases of the lower and upper chain are the same at the midpoint.
• Frequently palindromic.

Restriction Enzyme Activity

• Methylation of type II enzyme restriction sites affects DNA restriction.

Ends Generated by Restriction Enzymes

• Blunt Ends: Created by enzymes like AluI.
• Sticky Ends: Created by enzymes like EcoRI.

Relationships Between Enzymes

• Compatible Ends: Generated by different enzymes (e.g., BamHI, BglII, Sau3A).
• Isoschizomers: Recognize the same target sequence (e.g., SmaI, XmaI).
• Neoschizomers: Recognize the same sequence but cut at different places (e.g., SmaI, XmaI).

Restriction Reaction Components

• DNA, enzyme, buffer.
• Buffer contains pH, ionic strength (NaCl or potassium acetate), Mg^{2+} ions.
• Reducing agent: DTT or 2-mercaptoethanol.
• Add enzyme at the end to avoid undesired activity.

Stopping Restriction Reaction

• Heat inactivation at 70-80°C.
• Adding EDTA (chelates Mg^{2+}).
• DNA extraction to remove the enzyme.

Star Activity

• Occurs under non-optimal conditions; enzyme cuts at incorrect sites.
• Caused by high glycerol concentration.

Double Digestions

• Compatible temperatures and buffers: Digest simultaneously.
• Compatible buffer, incompatible temperature: Sequential reactions.
• Incompatible buffer: Perform separate reactions with DNA purification in between.

Enzymes for DNA Binding In Vitro

• DNA Ligases
• Topoisomerases
• Recombinases

DNA Ligases

• In vivo: Repair breaks in DNA strands.
• In vitro: Bind DNA strands together.
• T4 DNA ligase: Derived from T4 phage; requires ATP and Mg^{2+} ions; binds blunt or cohesive ends and RNA.
• E. coli DNA ligase: Requires NAD^+ and Mg^{2+} ions; binds only dsDNA cohesive ends.

DNA Ligase Mechanism

1. Enzyme-AMP complex binds to a nick with 3'OH and 5'P groups.
2. AMP reacts with the phosphate group.
3. 3'OH group attacks, forming a phosphodiester bond.

Ligases: Endpoint Modification

• Cohesive to Blunt Ends:
  • Trimming: Use polymerases with 3'-5' exonuclease activity.
  • Filling: Incubate with Klenow and dNTPs.
    • Blunt to Cohesive Ends:
    • Linkers
    • Adaptors
    • Homopolymer Tailing

Topoisomerase Ligation

• Vaccinia virus DNA topoisomerase cuts and joins DNA (TOPO-TA cloning).
• Vectors (pCR-TOPO) have 3'-T ends.
• Ligase-free; DNA topoisomerase performs ligation.

Recombinases

• Site-specific recombinases catalyze reciprocal exchange of dsDNA at specific sequences.
• Examples: integrase (int) of phage l(GATEWAY), cre protein of phage P1 (CRE/LOX).