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:
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- Typical LAMP Protocol (M0538)
- Typical LAMP Protocol (M0275)
- Typical LAMP Protocol (M0374)
- WarmStart LAMP Kit (DNA & RNA) Protocol (E1700)
- WarmStart Colorimetric LAMP 2X Master Mix Typical LAMP Protocol (M1800)
- One-Step RT-HDA (Reverse Transcription tHDA)
- One-Step tHDA (thermostable HDA)
- One-Step qRT-HDA (Real-time quantitative RT-HDA)
- One-Step qHDA (Real-time quantitative tHDA)
- Two-Step qHDA (Real-time quantitative tHDA)
- Two-Step qRT-HDA (Real-time quantitative RT-HDA)
- Two-Step RT-HDA (Reverse Transcription tHDA)
- Two-Step tHDA (thermostable HDA)
- A-Tailing with Klenow Fragment (3'-->5' exo-)
Anatomy of a Polymerase - How Structure Effects Function
- 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
- 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
- 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
- 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
- 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
- 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
- Tanner NA, Evans TC Jr. 2014. Loop-mediated isothermal amplification for detection of nucleic acids Curr Protoc Mol Biol. 105, PubMedID: 24510439, DOI:
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?
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.
Isothermal amplification generates many copies of a target sequence in a short period of time, at a constant temperature. Learn more about isothermal amplification.