Rapid detection of CALR type 1 and type 2 mutations using PNA-LNA clamping loop-mediated isothermal amplification on a CD-like microfluidic chip

Cao, Guikang; Kong, Jilie; Xing, Zhifang; Tang, Yigui; Zhang, Xinju; Xu, Xiu-Lian; Kang, Zhihua; Fang, Xueen; Guan, Ming

doi:10.1016/j.aca.2018.04.022

Cited by 32 publications

(9 citation statements)

References 22 publications

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“…In particular, the DNA-binding protein MutS from T. aquaticus is of particular interest, which became the basis for the detection of nucleotide polymorphisms in one of the asymmetric methods of isothermal amplification [ 126 ]. Improvement of primer design algorithms and rational inclusion of modified nucleotides, such as LNA and PNA, in their sequence [ 127 , 128 ] can increase the storage stability of primers and lead to the emergence of more specific allele-specific LAMP methods.…”

Section: Discussionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification: From Theory to Practice

Ширшиков

Bespyatykh

2022

Russ J Bioorg Chem

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Increasing the accuracy of pathogen identification and reducing the duration of analysis remain relevant for modern molecular diagnostics up to this day. In laboratory and clinical practice, detection of pathogens mostly relies on methods of nucleic acid amplification, among which the polymerase chain reaction (PCR) is considered the “gold standard.” Nevertheless, in some cases, isothermal amplification methods act as an alternative to PCR diagnostics. Upon more than thirty years of the development of isothermal DNA synthesis, the appearance of loop-mediated isothermal amplification (LAMP) has enabled new directions of in-field diagnostics of bacterial and viral infections. This review examines the key characteristics of the LAMP method and corresponding features in practice. We discuss the structure of LAMP amplicons with single-stranded loops, which have the sites for primer annealing under isothermal conditions. The latest achievements in the modification of the LAMP method are analyzed, which allow considering it as a unique platform for creating the next-generation diagnostic assays.

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Section: Discussionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification: From Theory to Practice

Ширшиков

Bespyatykh

2022

Russ J Bioorg Chem

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“…Studies have shown that almost all MPNs are associated with either a JAK2 V617F mutation or changes in JAK2 signaling, either directly or indirectly (Nangalia et al, 2013). JAK2 drives CML by phosphorylating BCR‐ABL1 and triggers 50‒60% of ET with the myeloproliferative leukemia virus oncogene ( MPL ) (3%‒5%) and calreticulin ( CALR ) (30%‒40%) responsible for the rest of ETs (Cao et al, 2018; Gángó et al, 2018). Although CALR mutations have been identified as markers of MPNs, the underlying molecular process remains largely unrevealed, prompting us to further explore the mechanism of CALR ‐mediated ETs at the molecular level.…”

Section: Introductionmentioning

confidence: 99%

Unraveling the connection between calreticulin and myeloproliferative neoplasms via calcium signaling

Jaiswal

Wang

Zhu

et al. 2023

Cell Biology International

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Mutations in the form of insertions and deletions (INDEL) in the calreticulin gene lead to essential thrombocythemia (ET) which is characterized by the formation of thrombosis.However, the connection between calreticulin INDEL and ET remains largely elusive.Through combined molecular dynamics simulation, clustered regularly interspaced short palindromic repeats (CRISPR) and calcium imaging studies on the wild type and mutated isoforms of calreticulin, the mechanism underlying the calreticulin INDEL induced ET was investigated at the molecular level. Our results demonstrate that mutations in exon-9 could lead to significant conformational variations of calreticulin structure and thereby reducing its interaction with calcium ions due to decreased electrostatic contributions.The consequence of mutations on calreticulin's structural integrity was revealed by identifying the key residues and their roles in calcium binding. Furthermore, mutations implemented by CRISPR-Cas9 in exon-9 showed diminished calcium signaling in HEK-293T cells, which agree well with our in-silico findings. The current study might help in understanding the variations of molecular interactions between calreticulin's exon-9 and calcium ions during physiological and pathological conditions. The results might also provide useful information for designing novel therapeutic approaches targeting ET.

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“…In general, the treatment of MPNs using disease modifiers is further complicated, as the specific treatment depends on the individual mutation [ 16 ]. Diagnosis of MPNs is aided by the detection of the various associated mutations by polymerase chain reaction, sequencing, or other DNA-based methods [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ], but the detection of disease-associated mutations may be facilitated or aided by mutation-specific immunohistochemistry (IHC) [ 31 , 32 , 33 ], methods which supplement each other. Moreover, antibodies such as these are invaluable reagents for studying the properties of CRT in relation to MPN.…”

Section: Introductionmentioning

confidence: 99%

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

Mughal

Bergmann

Huynh

et al. 2022

IJMS

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Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

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Rapid detection of CALR type 1 and type 2 mutations using PNA-LNA clamping loop-mediated isothermal amplification on a CD-like microfluidic chip

Cited by 32 publications

References 22 publications

Loop-Mediated Isothermal Amplification: From Theory to Practice

Loop-Mediated Isothermal Amplification: From Theory to Practice

Unraveling the connection between calreticulin and myeloproliferative neoplasms via calcium signaling

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

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