Loop-mediated isothermal amplification of DNA
Tsugunori Notomi,Hiroto Okayama,Harumi Otawara-shi Masubuchi,Toshihiro Yonekawa,Keiko Watanabe,Nobuyuki Amino,Tetsu Hase +6 more
TLDR
A novel method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions that employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.Abstract:
We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.read more
Citations
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Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences
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Nucleic acid detection with CRISPR-Cas13a/C2c2
Jonathan S. Gootenberg,Omar O. Abudayyeh,Jeong Wook Lee,Patrick Essletzbichler,Aaron J. Dy,Aaron J. Dy,Julia Joung,Vanessa Verdine,Nina M. Donghia,Nichole M. Daringer,Catherine A. Freije,Catherine A. Freije,Cameron Myhrvold,Cameron Myhrvold,Roby P. Bhattacharyya,Jonathan Livny,Aviv Regev,Aviv Regev,Eugene V. Koonin,Deborah T. Hung,Pardis C. Sabeti,James J. Collins,Feng Zhang +22 more
TL;DR: A Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), is used to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA.
Journal ArticleDOI
Accelerated reaction by loop-mediated isothermal amplification using loop primers.
TL;DR: The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP, which should facilitate genetic analysis, including genetic diagnosis in the clinical laboratory.
Journal ArticleDOI
CRISPR-Cas12-based detection of SARS-CoV-2.
James Paul Broughton,Xianding Deng,Guixia Yu,Clare L Fasching,Venice Servellita,Jasmeet Singh,Xin Miao,Jessica Streithorst,Andrea Granados,Alicia Sotomayor-Gonzalez,Kelsey C. Zorn,Allan Gopez,Elaine Hsu,Wei Gu,Steve Miller,Chao Yang Pan,Hugo Guevara,Debra A. Wadford,Janice S. Chen,Charles Y. Chiu +19 more
TL;DR: The CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT–PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.
Journal ArticleDOI
Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products
TL;DR: An improved simple visual detection system for the results of the LAMP reaction that enables visual discrimination of results without costly specialized equipment should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
References
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Journal ArticleDOI
Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.
John C. Guatelli,Kristina Marie Whitfield,Deborah Y. Kwoh,Kevin J. Barringer,Douglas D. Richman,Thomas R. Gingeras +5 more
TL;DR: A target nucleic acid sequence can be replicated exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase, and this reaction accumulates cDNA and RNA copies of the original target.