Polymerases and Amplification Technologies
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  • Isothermal Amplification & Strand Displacement

    The Polymerase Chain Reaction (PCR) is a well-known approach and method to replicate a specific DNA sequence. PCR involves the iterative cycling of a reaction cocktail between different temperatures to achieve amplification. As routine as PCR is in molecular biology and molecular diagnostic laboratories, there are other methods of sequence-specific DNA amplification. These alternative approaches often do not require changing the reaction temperature and are, therefore, often referred to as sequence-specific isothermal amplification protocols. Isothermal amplification protocols are varied and thus have varied advantages. However, some common advantages are that isothermal techniques are extremely fast and they do not require thermocyclers.

    Four examples of sequence-specific isothermal DNA amplification technologies include:
    1. Behind the Paper: Visual Detection of Isothermal Nucleic Acid Amplification Using pH-sensitive Dyes

      One little proton could change the way scientists detect DNA amplification in the field and point-of-care settings. Nathan shares the details of his recent paper.

  • Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA. A strand-displacing DNA polymerase initiates synthesis, and 2 of the primers form loop structures to facilitate subsequent rounds of amplification. LAMP is rapid, sensitive, and amplification is so extensive that the magnesium pyrophosphate produced during the reaction can be seen by eye, via the changes in turbidity making LAMP well-suited for field diagnostics.
  • Strand displacement amplification (SDA) relies on a strand-displacing DNA polymerase, typically Bst DNA polymerase, Large Fragment (NEB #M0275) or Klenow Fragment (3'→5' exo-), to initiate replication at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. SDA is typically used in clinical diagnostics.
  • Helicase-dependent amplification (HDN) employs the double-stranded DNA unwinding activity of a helicase to separate strands, enabling primer annealing and extension by a strand–displacing DNA polymerase. Like PCR, this system requires only two primers. HDA has been employed in several diagnostic devices and FDA-approved tests.
  • Nicking enzyme amplification reaction (NEAR) employs a strand-displacing DNA polymerase initiating replication at a nick created by a nicking enzyme, rapidly producing many short nucleic acids from the target sequence. This process is extremely rapid and sensitive, enabling the detection of small target amounts in minutes. NEAR is already being used commonly used for pathogen detection in clinical and biosafety applications.
  • Thermophilic Helicase-Dependent Amplification (tHDA) is a novel method for isothermal amplification of nucleic acids. Like PCR, the tHDA reaction selectively amplifies a target sequence defined by two primers. However, unlike PCR, tHDA uses a helicase enzyme, rather than heat, to separate dsDNA. This allows DNA amplification without the need for thermocycling. The tHDA reaction can also be coupled with reverse transcription for RNA analysis.

    The IsoAmp® II Universal tHDA Kit (NEB #H0110) can be used to amplify and detect short DNA sequences (70 bp - 120 bp) at a constant temperature. The kit can be used with a variety of templates, including microbial genomic DNA, viral DNA, plasmid DNA, and cDNA. When optimized primers and buffer are used, even a single copy of target DNA can be amplified by tHDA and detected by agarose gel electrophoresis.

    The IsoAmp II Universal tHDA Kit is based on a second-generation thermophilic Helicase-Dependent Amplification platform. The reactions supported by IsoAmp II Universal tHDA Kit include tHDA, reverse transcription HDA (RT-HDA), real-time quantitative HDA (qHDA) and real-time quantitative RT-HDA (qRT-HDA), from a single reaction buffer. The IsoAmp II Universal tHDA Kit contains an enzyme mix, dNTP solution and buffer, allowing flexibility in reaction setup. A control template and a set of specific primers are supplied for the positive control reaction.

    Recommended storage conditions for the tHDA Kit are -20°C for storage shorter than 6 months and -80°C for storage greater than 6 months. Repeated freeze-thaw cycles should be avoided as this may decrease the performance of the kit.

    Loop-mediated Isothermal Amplification (LAMP)
    Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA. A strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification.

    Overview of Strand Displacement Amplification (SDA)

    IsoAmp® is a registered trademark of New England Biolabs, Inc.

    DNA Polymerase Selection Chart

    NEB offers a guidelines for choosing the correct DNA polymerase for your application by providing a list of specific properites.
    Several factors govern which polymerase should be used in a given application, including: 

    Template/product specificity: Is RNA or DNA involved? Is the 3´ terminus at a gap, nick or at the end of the template? 

    Removal of existing nucleotides: Will the nucleotide(s) be removed from the existing polynucleotide chain as part of the protocol? If so, will they be removed from the 5´ or the 3´ end? 

    Thermal stability: Does the polymerase need to survive incubation at high temperature or is heat inactivation desirable? 

    Fidelity: Will subsequent sequence analysis or expression depend on the fidelity of the synthesized products?