Unit 1 - Genetic Engineering Flashcards

Theoretical Foundations of Genetic Engineering

• Introduction to genetic engineering.
• Basic laboratory techniques.
• Basic enzymology for DNA manipulation in vitro.
• Molecular Cloning I, II, and III: Basic and Cellular Cloning Systems and Cloning Methods.
• Advanced vectors in bacteria and other organisms.
• Gene Libraries.
• Expression of cloned genes.
• Modification of DNA sequences: mutagenesis and gene editing.
• Synthetic Biology.

Basic Enzymology

• In vitro DNA modification, cutting, and binding.
• Enzymes perform functions in vitro (laboratory tube).

DNA Modifying Enzymes

• Nucleases: cut, shorten, or degrade nucleic acid molecules.
• Polymerases: copy DNA molecules.
• Other DNA modifying enzymes: add or remove radicals or chemical groups.

Nucleases

• Degrade DNA by breaking phosphodiester linkages.
• Exonucleases: remove nucleotides from the ends of the chain.
• Endonucleases: break phosphodiester bonds internally.
• Examples of Exonucleases:
  • Bal31: degrades both 3' and 5' termini of duplex DNA.
  • Exonuclease III: removes nucleotides from only one strand.
• Lambda Exonuclease: Highly processive 5'-3' exonuclease that catalyzes the removal of 5' mononucleotides from duplex DNA. The preferred substrate is 5'-phosphorylated double stranded DNA
• Examples of Endonucleases:
  • S1 nuclease: Short ssDNA
  • DNase I: Cutting dsDNA and ssDNA; used in RNA extraction.
  • Restriction endonucleases.
• Other Nucleases:
  • RNAse A: Degrades ssRNA; used in DNA extraction.
  • RNAse H: Removes RNA strand after cDNA synthesis in RT-PCR.

Polymerases

• Synthesize new DNA or RNA strands using a template.
• Types:
  • 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.
    • Applications: RT-PCR, radiolabeling DNA fragments.
  • Klenow Fragment:
    • Lacks 5'-3' exonuclease activity but maintains 3'-5' activity (proofreading).
    • Applications: Filling in nicks or holes in dsDNA, filling dsDNA ends with 5' overhangs.
    • A variant also lacks 3'-5' exonuclease activity and is used for random priming.
  • Reverse Transcriptase (RT):
    • 5'-3' DNA polymerase activity on RNA template.
    • Synthesizes cDNA from single-stranded RNA or DNA.
  • RNA Polymerases (DNA-dependent):
    • Phage RNA polymerase (T7, T3, SP6): in vitro RNA synthesis.
    • RNA polymerase from E. coli: in vitro transcription with suitable promoters.
    • RNA polymerase II (wheat): study of transcription in eukaryotes.

Other DNA Modifying Enzymes

• Alkaline phosphatase:
  • Removes the phosphate group from a free 5' DNA end; prevents vector religation during cloning.
• Polynucleotide kinase:
  • Adds a phosphate group to a free 5' end; used for radioactive labeling and phosphorylation of oligonucleotides.
• Terminal Transferase:
  • Adds terminal dNTPs to the 3'OH end of dsDNA or ssDNA.
  • Applications: Creation of tails in a process called homopolymer tailing, radioactive labeling.
• Other modifications: Methylase, Poly(A)-polymerase, etc.

Restriction Endonucleases

• Bacterial protection against bacteriophages.
• Work with DNA methylases to protect bacterial DNA.
• Type II restriction enzymes are used in genetic engineering.
• Nomenclature: 3-4 letters in italics (e.g., EcoRI).
• Recognition sites:
  • 4-8 base pairs.
  • Symmetrical and frequently palindromic.
• Can generate blunt or cohesive (sticky) ends.
• Relationships between enzymes:
  • Compatible ends.
  • Isoschizomers (recognize the same target sequence).
  • Neoschizomers (recognize the same sequence but cut in different places).

Restriction Reaction Components

• DNA.
• Enzyme.
• Buffer: pH, ionic strength, Mg ions, reducing agent.
• Incubation temperature.
• Sometimes Bovine Serum Albumin (BSA).

Star Activity

• If reaction conditions are not optimal, the enzyme can cut at wrong sites.
• Double Digestions: Use compatible buffers and temperatures when digesting with two restriction enzymes.

DNA Binding In Vitro

Ligases

• In vivo: Repair of breaks in DNA strands.
• In vitro: Bind DNA strands together.
• Types:
  • T4 DNA ligase: Binds any DNA with blunt or cohesive ends; also binds RNA.
  • E. coli DNA ligase: Binds only dsDNA cohesive ends (no RNA).

Modification of Endpoints

• Change from cohesive to blunt ends:
  • Trimming the protruding end using polymerases with 3'-5' exonuclease activity.
  • Filling in the single-stranded region using Klenow and dNTPs.
• Change from blunt ends to cohesive ends using linkers, adaptors or homopolymer tailing.

Linkers

• Used for cloning microRNAs and regulatory elements.

Adaptors

• Used for cloning cDNA from microRNAs or other small RNAs and 3'-RACE.

Homopolymer Tailing

• Rarely used due to the availability of restriction enzymes.

Topoisomerase Ligation

• DNA topoisomerase from Vaccinia virus performs simultaneous DNA cutting and joining.
• TOPO-TA cloning kit system (Invitrogen) for cloning PCR products.

Recombinases

Site-specific RECOMBINASES (site-specific RECOMBINASES) are used instead of ligases: They catalyze the reciprocal exchange of 2 dsDNA molecules, when at specific sequences are present in these molecules

• Examples:
  • Integrase (int) of phage l: commercial system GATEWAY (Invitrogen)
  • Cre protein cre phage P1: CRE/LOX system