WEEK 15: GENETIC APPLICATION

• biotechnology

  • manipulation of DNA
  • the use or alteration of cells or biological molecules for specific applications (i.e., products & processes)
  • genetic engineering or genetic modification
• transgenic

  • multicellular organisms that harbor DNA from other species; their DNA is called recombinant DNA
• 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
• recombinant dna technology

  • 3 Components in manufacturing recombinant DNAmolecules:
  • Restriction enzymes
  • Cloning vectors
  • Bacteriophage (i.e., bacteriophage λ)
  • 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)
  • Recipient cells
• RESTRICTION ENZYMES/ENDONUCLEASES

  • Recognize a specific base sequence in a DNA molecule and cut near or within the sequence – usually a palindrome
• POLYMERASE CHAIN REACTION

  • Kary Mullis - invention of the polymerase chain reaction method
  • Best known and most widely applied
  • PCR components:
  • Thermostable DNA polymerase – Taq polymerase
  • 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
  • Buffer – Mg 2+and KCl
• PCR STEPS

  1. Denaturation - 90-96 celsius and 20-60 seconds
  2. Annealing - 50-70 celsius and 20-90 seconds
  3. Extension - 68-75 celsius and 10-60 seconds
• MODIFICATIONS IN POLYMERASE CHAIN REACTION

  • NESTED PCR
  • 2 pairs of primers + single target
  • 2 separate PCR runs
  • MULTIPLEX PCR
  • Amplifying more than 1 target simultaneously in the same solution –consensus primers
  • REVERSE TRANSCRIPTASE PCR(RT-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)
  • Quantitative
  • Simultaneous amplification and detection (fluorescence signaling)