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DNA Amplification

Isothermal Amplification

The Polymerase Chain Reaction (PCR) is a well-known approach to amplify a specific DNA sequence. PCR involves the reiterative cycling of a reaction cocktail between different temperatures to achieve amplification. As routine as PCR is in the molecular biology and molecular diagnostic laboratory, 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 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:
  •  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 or Klenow Fragment (3’-5’ exo–), to initiate 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 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 detection of small target amounts in minutes. NEAR is commonly used for pathogen detection in clinical and biosafety applications.


    • 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)

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    FAQs for Isothermal Amplification
    Protocols for Isothermal Amplification
      Publications related to Isothermal Amplification
    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:
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    Videos

    1. LAMPoverview_Video_thumb

      Loop Mediated Isothermal Amplification (LAMP) Tutorial

      Did you know that this isothermal amplification method can be performed in as little as 5-10 minutes with limited resources? Get a quick overview on how LAMP works in this animation.

    2. NathanTanner_BehindPaper_thumb_282x210

      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.

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      NEB TV Ep. 15 – Applications of Restriction Enzymes

      These days, restriction enzymes are being used in many more applications other than cloning. Learn more in this episode of NEB TV.

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