Week 15

• Biotechnology


  Manipulation of DNA // The use or alteration of cells or biological molecules for specific applications (i.e., products & processes); need to change/address; genetic engineering/genetic modification
• Transgenic


  Multicellular organisms that harbor DNA from other species; their DNA is called Recombinant DNA; examples are Crops like BT Talong, Golden Rice wherein they have components that they don't own
• Recombinant DNA Technology


  Adds genes from one type of organism to the genome of another

  
  

  The 1st gene modification biotechnology

  
  

  Was initially done with bacteria to produce peptides and proteins - drugs

  
  

  Also known as "gene cloning" - making many copies of a specific DNA Sequence
• EcoRI/Escerichia coli RY13


  Treated with restriction enzymes to cut plasmid -> sticky ends/blunt ends -> Heat Shock treatment (Transformation) -> rDNA

  
  

  Common/first use restriction enzyme
• Plasmid


  Extrachromosomal DNA found in bacteria
• Recombinant DNA Technology


  3 components in manufacturing recombinant DNA molecules:

  
  

  1. Restriction Enzymes (make cuts/molecular scissors)

  
  

  2. Cloning vectors

  - Bacteriophage (i.e., bacteriophage lambda)

  - Plasmid (i.e., pBR322, pUC19)

  - Cosmid - hybrid vector combining features of plasmids and bacteriophages

  
  

  - Bacterial Artificial Chromosome (BAC)

  - Yeast Artificial Chromosome (YAC)

  - Human Artificial Chromosome (HAC)

  = These three are used for whole genome sequencing/large sequences

  
  

  3. Recipient Cells (i.e. bacteria)
• Ligase


  Glue/Seal Sequence
• Restriction Enzymes/Endonucleases


  Recognize a specific base sequence in a DNA molecule and cut near or within the sequence - usually a palindrome
• BamHI/Bacillus amyloliquefaciens H

  Second most common restriction enzyme
• Bacteriophage (Cloning Vectors)


  Plaques; COS - apply cohesive ends to package into phage particles
• Plasmid (Cloning Vectors)


  Insert antibiotic resistance
• Cosmid (Cloning Vectors)


  Combination of plasmid and bacteriophage
• Atrial Natriuretic Peptide (Drug)


  Dilates blood vessels, promotes urination (Use)
• Colony-Stimulating Factors (Drug)


  Help restore bone marrow after marrow transplant; restore blood cells following cancer chemotherapy
• Deoxyribonuclease (DNase - Drug)


  Thins secretions in lungs of people with cystic fibrosis
• Epidermal Growth Factor (Drug)


  Accelerates healing of wounds and burns; treats gastric ulcers (Use)
• Erythropoietin (EPO - Drug)


  Stimulates production of red blood cells in cancer patients (Use)
• Factor VIII (Drug)


  Promotes blood clotting in treatment of hemophilia A (Use)
• Glucocerebrosidase (Drug)


  Corrects enzyme deficiency in Gaucher disease (Use)
• Human Growth Hormone (Drug)


  Promotes growth of muscle and bone in people with very short stature due to hormone deficiency (Use)
• Insulin (Drug)


  Allows cells to take up glucose in treatment of type 1 diabetes (Use)
• Interferons (Drug)


  Treat genital warts, hairy cell leukemia, hepatitis B and C, Kaposi sarcoma, multiple sclerosis
• Interleukin-2 (Drug)


  Treats kidney cancer recurrence (Use)
• Lung Surfactant Protein (Drug)


  Helps lung alveoli to inflate in infants with respiratory distress syndrome (Use)
• Renin Inhibitor (Drug)


  Lowers blood pressure (Use)
• Somatostatin (Drug)


  Decreases growth in muscle and bone in pituitary gigantism (Use)
• Thrombin (Drug)


  Stops postsurgical bleeding (Use)
• Tissue Plasminogen Activator (Drug)


  Dissolves blood clots in treatment of heart attack, stroke, and pulmonary embolism (Use)
• Polymerase Chain Reaction


  Amplification Technique and Various changes in temperature

  
  

  Best known and most widely applied -> copy the replication process

  
  

  Components:

  - Thermostable DNA polymerase - Taq polymerase (Thermos aquaticus = bacteria)

  
  

  - Deoxynucleotides (Deoxyribonucleoside triphosphates/dNTPs)

  
  

  - DNA strand (target sequence to be amplified) / Template DNA

  
  

  - Primers (pair of oligonucleotides - complementary to opposite strands) - forward and reverse primers -> to accommodate both lagging/leading strands

  
  

  - Buffer - Mg2+ and KCl
• PCR Machine


  It is a thermal cycler
• Kary Mullis


  Invention of the polymerase chain reaction method
• PCR amplifies a specific nucleic acid sequence


  Many types of viruses cause upper respiratory infections. It illustrates how PCR is used on secretions to identify an adenovirus, which has double-stranded DNA as its genetic material. Influenza viruses have RNA as the genetic material, and a different form of PCR, called reverse transcription PCR, is used to analyze them

  
  

  2^N - N - No. of Cycles (every cycle, 2 is produced)
• Denaturation


  1st step in PCR Cycle

  
  

  Unwinds single stranded from double stranded (1 cycle - routine is 30-40 cycles)

  
  

  90-96 degrees (Temperature)

  
  

  20-60 seconds (Time)
• Annealing


  2nd step in PCR Cycle

  
  

  Primer attaches

  
  

  50-70 degrees (Temperature)

  
  

  20-90 seconds (Time)
• Extension


  Last step in PCR Cycle

  
  

  Create a new copy of sequence

  
  

  68-75 degrees (Temperature)

  
  

  10-60 seconds (Time)
• Nested PCR (Modifications in PCR)

  2 pairs of primers + single target

  2 separate PCR runs
• Multiplex PCR (Modifications in PCR)


  Amplifying more than 1 target simultaneously in the same solution - consensus primers (infectious disease; simultaneously)
• Reverse Transcriptase PCR (RT-PCR = Modifications in PCR)


  Starting material: RNA

  
  

  First converted to dsDNA (better template for amplification) - Reverse transcriptase (RT)

  
  

  RNA:DNA complex/hybrid >> cDNA (complementary DNA)
• Real-Time PCR (qPCR = Modifications in PCR)


  Quantitative

  
  

  Simultaneous amplification and detection (fluorescence signaling)
• Reverse Transcription PCR


  Convert RNA then amplification