تقنيات

• Sequencing


  The technology that determines each of the four nucleotides (A, G, T, C) in the right position in DNA strand
• Types of sequencing technology

  • Sanger chain termination sequencing
  • Maxam-Gilbert chemical degradation method
  • Whole genome sequencing (WGS)
  • Whole exome sequencing
  • Next generation sequencing (NGS)
• Sanger chain termination sequencing

  1. Termination of one nucleotide at one end of DNA strand by using a nitrogen base analogue but lacks to OH at the third position of deoxyribose and called "2,3 dideoxynucleotide triphosphates (ddNTPs)" preventing DNA polymerase from adding the nitrogen base to the growing strand during PCR

  2. Four tubes containing template DNA, the four nucleotides (dNTPs; A, G, C, T), primers, Taq DNA polymerase, and a low concentration of ddNTPs assigned for each of the nucleotides to stop the reaction

  3. Resulting sequences are different in lengths but resemble in sequences except in one nucleotide

  4. Visualized either by gel or capillary electrophoresis
• Maxam-Gilbert chemical degradation method

  1. Denaturation of the DNA duplex into one single strand and removed of phosphate group from 5´ end by alkaline phosphatase and replace it by radiolabeled P32 by kinase

  2. PCR is conducted to copy millions of the labelled DNA strand

  3. Four tubes each one contains the radiolabeled template a chemical that is assigned for each base to cleave

  4. Different DNA strand cuts will be generated according to each base cleaved

  5. Cuts are all subjected to electrophoresis and visualized via autoradiograph
• Maxam-Gilbert chemical degradation method

  • Laborious, toxic, difficulties appear when sequencing more than 500 bp, and cleavage errors
• Whole genome sequencing (WGS)

  The entire genome of any organism is sequenced
• Whole genome sequencing (WGS)

  • Detection of mutations, determining the genetic disorders, detecting the outbreaks
  • Produces a large data information in short time, screens total variants either major or minor, and also it sequences multiple samples at time
• Whole exome sequencing


  The technology is used to sequence the total exons of the human which code for proteins
• Whole exome sequencing


  • Very efficient method to detect the mutations
• Next generation sequencing (NGS)

  Sequences millions of DNA fragments simultaneously that summarizes the time consuming Sanger sequencing for human genome for only a day or two
• Next generation sequencing (NGS)

  1. Preparing a library

  2. Sequencing

  3. Data analysis
• Next generation sequencing (NGS)

  • Bioinformatics are used to collect all these fragments and mapping them comparing to reference human genome
  • All the human genome is sequenced multiple times so as to detect the unexpected variants, or sequencing only coding genes (whole exome)
  • Reads<400bp and the reads number 1 million-1 billion reads /run
• Main types of NGS

  • Pyrosequencing (454)
  • Reversible terminator sequencing (Illumina)