Lecturer 1 intro to mg

• Molecular genetics


  Sub-discipline of genetics that studies the structure and function of genes at a molecular level, including DNA structure, RNA structure, gene expression, gene mutation, gene editing and gene therapy

  
• Molecular genetics is the study of the structure and function of genes at a molecular level
• Central dogma


  • The flow of genetic information from DNA to RNA to protein

  
• DNA


  A long molecule with a double helix structure, found in all living organisms except plant viruses. Components: sugar molecule (deoxyribose), phosphoric acid, and nitrogenous bases (Adenine, Guanine, Cytosine, Thymine)

  
• RNA


  A single-stranded molecule found in the cytoplasm and nucleolus. Components: sugar molecule (ribose), phosphoric acid, and nitrogenous bases (Adenine, Guanine, Cytosine, Uracil)

  
• Gene expression


  The process of transcription and translation which leads to the production of proteins

  
• Transcription


  The process where DNA is used as a template to produce mRNA in the nucleus

  
• Translation


  The process where mRNA is used as a template to produce proteins in the cytoplasm

  
• Forward genetics


  • Identifying mutations and linking them to a certain phenotype
• Reverse genetics


  • Inducing mutations to study the phenotype
• Gene mutations


  Changes in the DNA of an organism, can be spontaneous or induced
• Gene therapy


  The process of treating or alleviating diseases by genetically modifying the cells of an affected person, to improve the function of a protein/gene/cellular metabolism
• Gene therapy examples


  • CRISPR, sickle-cell anemia, immunotherapy
• Classic gene therapy


  • Delivering a gene via a modified virus or vector to target cells to achieve optimal expression of a new, introduced gene

  
• Non-classic gene therapy


  • Inhibiting gene expression related to pathogenesis, or correcting a genetic defect to restore normal gene expression
• In vivo gene transfer


  • Genes are directly transferred into body cells, via viruses such as adenoviruses and retroviruses

  
• Ex vivo gene transfer


  • Cells are cultured outside the body, genes are transfected into the cells, then the transformed cells are expanded and introduced back to the patient

  
• How gene therapy works

  A normal gene is inserted into the genome to replace an abnormal disease-causing gene, using a vector to deliver the therapeutic gene to the patient's target cells

  
• Steps in gene therapy

  Isolate healthy genes with expression control sequence, incorporate gene on a carrier/vector as an expression cassette, deliver vector to target cells

  
• Gene therapy using adenovirus vector


  • Adenovirus is used as a vector to deliver the therapeutic gene to target cells

  
• Basic techniques in molecular genetics

  • Amplification
  • Separation and detection
  • Expression

  
• Amplification


  Techniques like PCR and cloning of DNA in bacteria to make copies of DNA fragments

  
• PCR (Polymerase Chain Reaction)


  A molecular technique that amplifies/copies small segments of DNA using Taq polymerase

  
• Materials required for PCR

  • Two primers (single stranded DNA)
  • DNA polymerase
  • Deoxynucleotides (dNTPs)
  • Target DNA fragment
  • Buffer solution
  • Divalent cations (Magnesium ion)
  • Monovalent cation (Potassium ion)
• Principles of PCR


  Repeated cycles of DNA denaturation, primer annealing, and DNA synthesis to exponentially amplify target DNA

  
• Advantages of PCR


  • Much faster using vectors
  • Small amount of DNA required
• Disadvantages of PCR


  • To synthesis Need to know sequence flanking DNA regions of interest to synthesize primers
  • Only short DNA fragments (<5-10kb) can be amplified

  
• RT-PCR (Reverse Transcriptase PCR)


  Technique that converts RNA to DNA using reverse transcriptase, then amplifies the DNA

  
• Reverse PCR


  Uses Tth polymerase which has both DNA polymerase and reverse transcriptase activities, allowing cDNA synthesis from mRNA followed by PCR amplification

  
• Restriction enzyme


  A protein that binds to DNA at a specific palindromic sequence and makes a double-strand excision, useful in RFLP, cloning, and more

  
• Vectors


  Used to transfer human genes into bacteria, common vectors are plasmids. A vector carrying a foreign gene is called a chimeric/hybrid/ recombinant DNA

  
• Expression vector


  A vector carrying a foreign gene which is expressed and translated into protein

  
• Cloning DNA in bacteria

  Process of creating an identical copy of a DNA fragment by inserting it into a cloning vector, transforming into E. coli, and allowing the E. coli to grow and divide, replicating the recombinant DNA

  
• Cell culture


  Cells grown in an artificial and controlled environment, can be frozen to preserve for future use (cryopreservation)
• DNA and mRNA isolation

  Breaking open cells and separating the nucleic acids from other cellular components. mRNA is more easily degraded compared to DNA.
• Terminology in molecular genetics

  • Gene mapping
  • Linkage analysis
  • Microsatellite markers
  • Gene library
  • Gel electrophoresis
  • RFLP
  • Western blot
  • Fluorescence in situ hybridization (FISH)
  • DNA sequencing