Polymerase chain reaction (PCR)

• DNA sequencing

  • Enables mapping of species genomes
  • Identifies the unique genetic makeup of individuals
  • Human beings can sequence even small amounts of DNA
• How is DNA sequencing done ?

  The base sequences of a gene or a genome is determined by using free DNA nucleotides labeled with different fluorescent dyes and DNA polymerase to form complementary strands with single DNA strands. The base sequence of the synthesized DNA strands are read using a laser scanner which translates a specific colored fluorescent dye as a type of DNA base thus the whole process is automated
• Applications of sequencing genes and genomes

  • Sequence unknown bases of specific genes to clone normal genes for gene therapy
  • Genome sequencing for different species to compare base sequences to determine relatedness for drug testing or scientific research
  • Cloning of the wooly mammoth from its genomes obtained from fossils to revive endangered or extinct species
• Polymerase Chain Reaction (PCR)

  1. Technology used to carry out DNA replication artificially in the laboratory

  2. Used to amplify identical copies of DNA from trace samples exponentially
• Applications of PCR

  • Amplification of DNA from trace samples of foetal cells obtained by amniocentesis for pre-natal screening and diagnosis of genetic disorders
  • Amplification of DNA from body fluids such as blood, semen, saliva for crime solving and identification of criminals or victims
  • Amplification of DNA obtained from fossils for cloning of endangered or extinct organisms
• The PCR Protocol

  Denaturation (DNA Strand Separation): The DNA molecule is heated to separate complementary DNA strands to act as template strand. 2. Annealing (Primer Attachment): The temperature is lowered to allow
• Population size or reproductive cloning
• THE PCR PROTOCOL
• PCR Protocol - Denaturation


  The DNA molecule is heated to separate complementary DNA strands to act as template strand
• PCR Protocol - Annealing

  The temperature is lowered to allow primers to bind to the DNA strands and to allow the DNA strands to be separate and act as templates for DNA replication. Primers are short sequences of DNA or RNA nucleotides synthesized artificially bind to the beginning on opposite site of both template DNA strands
• PCR Protocol - Extension

  The temperature is raised slightly, a thermostable DNA polymerase (also known as Taq DNA polymerase is isolated from bacteria living in hot springs) is added to complete the synthesis of a new DNA strand using free DNA nucleotides
• Materials required for PCR

  • Sample of template DNA (tissues or fluid), primers, Taq DNA polymerase, synthetic free DNA nucleotides, PCR machine
• Materials placed in the PCR tubes

  • Template DNA, Short primer, Taq DNA polymerase, Free DNA nucleotides
• Sequence of steps in the PCR protocol

  • Step 1 – Denaturation (DNA strand separation), Step 2 – Annealing (Primer attachment Step), Step 3 – Extension (DNA strand synthesis), Step 4 - Repeat step 1-3 again (exponential amplification Step)