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AAT Bioquest

What are the different techniques commonly used to amplify DNA fragments before sequencing?

Posted February 20, 2024


Answer

The three types of techniques used to amplify DNA fragments include: bridge amplification, DNA nanoball generation, and emulsion PCR

  1. Emulsion PCR utilizes the fact that PCR components, including template DNA, nucleotides, polymerase and primers are water-soluble but not oil-soluble. In this technique, DNA molecules are amplified within physically isolated water-in-oil droplets. Both GenapSys and Ion Torrent sequencing platforms utilize this technique to facilitate the amplification of DNA in a controlled manner. 
  2. The DNA nanoball generation, unlike PCR amplification, involves cloning a library of DNA fragments into a retroviral vector resulting in the production of circular DNA structures. Fewer copies of DNA are generated compared to bridge amplification or emulsion PCR, with only 300 copies produced from each original fragment. 
  3. In bridge amplification, DNA molecules undergo repetitive replication on a glass flow cell that has complementary oligonucleotides. Bridge amplification is inefficient for clonal amplification, producing only about 1,000 copies of the original molecule after 35 cycles. Additionally, there is a significant risk of template strands hybridizing with each other instead of annealing to a new primer site on the flow cell.
Additional resources

Amplifying DNA - The Polymerase Chain Reaction

Sanger Sequencing

MagaDye™ 535-ddGTP