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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 themocyclers.

Four examples of sequence-specific isothermal DNA amplification technologies include:
  • 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, 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 (HDA) 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.

  • LAMP_thumb
    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.

    SDA_workflow
    Overview of Strand Displacement Amplification (SDA)

    IsoAmp® is a registered trademark of BioHelix Corp.


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    FAQs for Isothermal Amplification & Strand Displacement

    Protocols for Isothermal Amplification & Strand Displacement

      Publications related to Isothermal Amplification & Strand Displacement

    1. Manabu Nemoto, Yoshinori Morita, Hidekazu Niwa, Hiroshi Bannai, Koji Tsujimura, Takashi Yamanaka, Takashi Kondo 2015. Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification. J Virol Methods. 215-216, PubMedID: 25682750, DOI: 10.1016/j.jviromet.2015.02.001
    2. Sanchita Bhadra, Yu Sherry Jiang, Mia R Kumar, Reed F Johnson, Lisa E Hensley, Andrew D Ellington 2015. Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). PLoS One. 10, PubMedID: 25856093, DOI: 10.1371/journal.pone.0123126
    3. Trisadee Khamlor, Petai Pongpiachan, Rangsun Parnpai, Kanchana Punyawai, Siwat Sangsritavong, Nipa Chokesajjawatee 2015. Bovine embryo sex determination by multiplex loop-mediated isothermal amplification. Theriogenology. 83, PubMedID: 25542460, DOI: 10.1016/j.theriogenology.2014.11.025
    4. DoKyung Lee, Eun Jin Kim, Paul E Kilgore, Soon Ae Kim, Hideyuki Takahashi, Makoto Ohnishi, Dang Duc Anh, Bai Qing Dong, Jung Soo Kim, Jun Tomono, Shigehiko Miyamoto, Tsugunori Notomi, Dong Wook Kim, Mitsuko Seki 2015. Clinical Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Neisseria meningitidis in Cerebrospinal Fluid. PLoS One. 10, PubMedID: 25853422, DOI: 10.1371/journal.pone.0122922
    5. Mohammad Reza Allahyar Torkaman, Kazunari Kamachi, Vajihe Sadat Nikbin, Masoumeh Nakhost Lotfi, Fereshteh Shahcheraghi 2015. Comparison of loop-mediated isothermal amplification and real-time PCR for detecting Bordetella pertussis. J Med Microbiol. 64, PubMedID: 25596118, DOI: 10.1099/jmm.0.000021
    6. Aongart Mahittikorn, Hirotake Mori, Supaluk Popruk, Amonrattana Roobthaisong, Chantira Sutthikornchai, Khuanchai Koompapong, Sukhontha Siri, Yaowalark Sukthana, Duangporn Nacapunchai 2015. Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP). PLoS One. 10, PubMedID: 25822175, DOI: 10.1371/journal.pone.0120997
    7. Tanner NA, Evans TC Jr. 2014. Loop-mediated isothermal amplification for detection of nucleic acids Curr Protoc Mol Biol. 105, PubMedID: 24510439, DOI:

    DNA Polymerase Selection Chart

    DNA_Polymerase_Selection_thumb
    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?

    Legal Information

    This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

    While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

    For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

    This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.