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• Gene sequencing


  A specific type of DNA sequencing that determines the order of nucleotides in a particular gene

  
• Gene


  • A segment of DNA that contains the instructions necessary to form and maintain the body
  • Serves as a template for protein synthesis, which plays important roles in various biological processes
• Gene sequencing process


  1. Isolate the DNA containing the target gene

  2. Amplify it through a process such as polymerase chain reaction (PCR)

  3. Determine the gene's nucleotide sequence

  
• Applications of gene sequencing

  • Identify mutations, variants and other genetic factors associated with disease
  • Contribute to the development of targeted therapies and medicine
  • Contribute to understanding of genetic diversity, evolution, and the basic mechanisms of life
• DNA


  • Can be twisted and untwisted
  • Opened up into two strands
  • Diced up into shorter pieces and spliced to other DNA
  • Anneals easily with changing temperatures

  
• General process of DNA sequencing

  1. Denature and clone DNA fragments

  2. Primer attachment

  3. Addition of dNTPs and ddNTPs

  4. Sequencing by gel electrophoresis

  
• Sanger sequencing method


  • Denature and clone DNA fragments
  • Primer attachment
  • Addition of dNTPs and ddNTPs
  • Sequencing by gel electrophoresis

  
• Maxam & Gilbert sequencing method

  • Preparation of single stranded DNA
  • Addition of radiolabeled phosphorus (32P) to 5' phosphate
  • Chemical termination process
  • Chemical treatment generates breaks at a small proportions of one or two of the four nucleotide bases in each of four reactions
  • Fragments arranged side by side in gel electrophoresis for size separation
  • Visualize fragments using X-ray film for autoradiography

  
• NGS workflow


  1. Library preparation

  2. Enrichment

  3. Sequencing and Analysis
• All NGS experiments follow the same generalized workflow