Molecular techniques 2

• gDNA


  Genomic DNA
• cDNA


  Complementary DNA

  
• cDNA libraries


  Collections of cDNA clones representing the mRNA molecules expressed in a cell or tissue

  
• cDNA synthesis


  1. RNA extraction

  2. First strand synthesis using oligo dT primers

  3. Second strand synthesis

  
• PCR


  Polymerase Chain Reaction - a technique that amplifies DNA thousands of times
• PCR


  1. Denaturation

  2. Primer annealing

  3. Polymerisation (extension)
• PCR


  • Copies a short DNA region multiple times
  • Reaction mixture contains template DNA, primers, dNTPs, DNA polymerase, salts and buffer
• PCR applications


  • Detect microorganisms at early stages of infection
  • Forensic medicine
  • Paternity testing
• DNA fingerprinting


  Technique that uses short tandem repeats (STRs) to create a unique DNA profile
• Sanger sequencing


  Method to determine the complete sequence of bases (A, T, C, G) in a DNA molecule

  
• Sanger sequencing


  1. Hybridisation

  2. Polymerisation

  3. Four sets of labelled reactions

  4. Polyacrylamide gel electrophoresis
• The Human Genome Project sequenced all 3.2 billion base pairs of the human genome in 2003
• Bioinformatics tools can be used to analyse DNA sequences, determine if they are known, and predict protein structure and function