MOLECULAR GENETICS

• Recombinant DNA


  Combining pieces of DNA from different organisms

  
• Gene cloning


  Making copies of DNA

  
• Genetic engineering


  Direct manipulation of genetic material for practical purposes
• Biotechnology


  Use of living organisms or their components to make products for us
• Cell-based Molecular Cloning


  1. Create and isolate a bacterial strain that replicates a copy of your gene

  2. Make DNA single stranded, allow double strands to re-form using a labeled version of your gene to make it easy to detect

  3. Make many copies of a specific region of the DNA

  
• Recombinant DNA Technology


  The technology of transferring a foreign gene from one organism to another
• Recombinant DNA Technology


  • Involves isolation and manipulation of DNA to make chimeric molecules
  • Chimeric molecule can be DNA, RNA, or protein containing sequences derived from two different species

  
• Recombinant DNA Technology


  • Genes, when inserted into a foreign host cell, can produce desired gene products
  • The DNA is inserted into another DNA molecule called 'vector'
  • Recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced
• Restriction enzymes


  They come from bacteria and recognize a particular pattern of DNA, often 4, 6 or 8 base pairs long, and then cut the DNA within this recognized sequence

  
• Sticky ends


  DNA cut by restriction enzymes has overhanging ends that can base pair with another piece of DNA

  
• Blunt ends


  DNA cut by restriction enzymes has no overhanging ends
• DNA ligase


  Enzyme that seals DNA into an opening created by a restriction enzyme

  
• Complementary DNA (cDNA) Synthesis

  1. Use oligo-dT primer to bind to poly-A tail

  2. Make the first DNA strand from the RNA using reverse transcriptase

  3. Remove the RNA with heat or alkali

  4. The 3' end spontaneously forms a small hairpin

  5. Extend the hairpin with DNA polymerase

  6. Cut the loop with S1 nuclease

  7. Attach synthetic linkers with DNA ligase and clone into a vector

  
• Plasmids


  Independently replicating DNA circles that can carry foreign DNA and replicate it in a host cell

  
• Making recombinant DNA in plasmids

  1. Plasmid is cut open with a restriction enzyme

  2. Foreign DNA is cut with the same enzyme

  3. The two DNAs are mixed, the sticky ends anneal together, and DNA ligase joins them

  4. The recombinant plasmids are transformed into E. coli using heat plus calcium chloride

  5. Cells carrying the plasmid are selected by adding an antibiotic

  
• Genomic library


  Collection of thousands of clones of bacteria containing recombinant plasmids that cover the entire genome

  
• cDNA library


  Collection of clones representing the expressed genes in a particular tissue
• Gel electrophoresis


  Technique to separate DNA based on the movement of DNA fragments from negative to positive, with smaller fragments travelling farther

  
• Major Analytic Techniques


  • Blotting and Hybridization Techniques
  • DNA sequencing
  • RFLP linkage analysis
  • DNA fingerprinting
  • Polymerase chain reaction

  
• Hybridization


  If DNA is made single stranded, it will pair up with another DNA (or RNA) with the complementary sequence, allowing detection of specific sequences

  
• Random primers labeling


  Use 32P-labeled dNTPs and short random oligonucleotides as primers to copy a DNA template and incorporate the label
• Southern Blot


  Technique to detect a specific DNA sequence in a complex mixture by cutting DNA with a restriction enzyme, running on a gel, transferring to a membrane, and hybridizing with a labeled probe

  
• Northern Blot


  Similar to Southern Blot but uses RNA instead of DNA, to detect and quantify RNA fragments
• Western Blot


  Technique to detect one protein in a mixture of proteins, giving information about the size of the protein
• Northern blotting


  A method for detecting and quantifying RNA expression
• Northern blotting procedure


  1. Prepare RNA samples and run RNA gel

  2. Northern transfer

  3. Probe preparation

  4. Prehybridization

  5. Hybridization

  6. Post-hybridization washing

  7. Signal detection
• Signal detection methods


  • Isotope
  • Non-isotope
• Northern blotting is similar to "Southern", not because it was invented by a person named "Northern"
• Northern blotting


  • RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions
  • The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe

  
• Western blotting


  A technique to detect and quantify specific proteins in a mixture
• Western blotting procedure


  1. Electrophoresing the protein sample

  2. Assembling the Western blot sandwich

  3. Transferring proteins from gel to nitrocellulose paper

  4. Staining of transferred proteins

  5. Blocking nonspecific antibody sites on the nitrocellulose paper

  6. Probing electroblotted proteins with primary antibody

  7. Washing away nonspecifically bound primary antibody

  8. Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitate

  9. Photographing the immunoblot

  
• Western blot analysis can detect one protein in a mixture of any number of proteins while giving information about the size of the protein
• Western blotting is used to confirm serious diagnosis suggested by ELISA such as HIV seroconversion
• Southwestern blotting


  A method for detecting protein-DNA interactions by applying a labeled DNA probe to a transfer membrane that contains a renatured protein

  
• Restriction Fragment Length Polymorphisms (RFLP)


  • The first DNA-based genetic mapping technique
  • Advantage: every individual has many variations in their DNA, so you don't need a special set of marker mutations. Also, the markers are co-dominant so you can accurately determine the genotype
  • Probe is a fragment of a cloned gene (labeled)
  • Genomic DNA is cut with a restriction enzyme
  • Polymorphic sites: the restriction site is present in some individuals but not in others (due to mutation)

  
• Polymerase Chain Reaction (PCR)


  • Used to amplify DNA
  • Discovered by Kary Mullis
  • Uses a heat-stable DNA polymerase, most commonly Taq Polymerase from the thermophilic microbe Thermus aquaticus

  
• Steps of PCR


  1. Denature DNA (94-96°C)

  2. Anneal (base pair) primers (50 – 65°C)

  3. Extend primers (72°C for polymerase to work)

  
• PCR has replaced cloning, particularly in the DNA sequencing, as it is faster and requires no vectors which can mutate
• PCR can be used forensically, to amplify tiny amounts of DNA from criminal evidence or to detect DNA sequences linked to inherited disorders
• DNA fingerprinting


  • Technique of using DNA fragment lengths to produce a distinctive banding pattern
  • Usually used to measure number of repeats of short sequences
  • Used in paternity suits, rape cases, corpse ID, etc.