Detection of mutations in mitochondrial DNA by droplet digital PCR

Sofronova, J. K.; Ilinsky, Yu. Yu.; Orishchenko, Кonstantin E.; Chupakhin, Evgeny; Lunev, Evgenii A.; Мазунин, И. О.

doi:10.1134/s0006297916100011

Cited by 9 publications

(10 citation statements)

References 26 publications

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“…The degree of heteroplasmy was subsequently determined with droplet digital polymerase chain reaction to quantify wild-type and m.3243A>G copy number. 1 The mitochondrial DNA mutation rate was as high as 90.8% in cardiomyocytes, compared with 29% in skin fibroblasts. Subsequently, endomyocardial biopsy was performed and revealed mitochondrial hyperplasia and morphological abnormalities of cristae (Figure).…”

Section: Discussionmentioning

confidence: 99%

A Man With Left Ventricular Hypertrophy

2022

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A man in his 40s with a 20-year history of bilateral sensorineural hearing loss was referred by his primary care physician for evaluation of left ventricular hypertrophy detected by echocardiography. The echocardiogram was performed because of an abnormal electrocardiogram demonstrating sinus tachycardia accompanied by high QRS voltage with a strain pattern in the precordial leads. He had no history of other illnesses. On physical examination, the patient was 154 cm tall and weighed 38 kg. The cardiovascular examination was unremarkable. Neurological examination showed mild cognitive impairment, poor coordination, and muscle weakness of the bilateral lower limbs. Blood test results revealed impaired glucose tolerance, hypothyroidism, and elevated levels of lactic acid (32.4 mg/dL) and pyruvate acid (1.91 mg/dL; to convert to micromoles per liter, multiply by 113.56). Review of the transthoracic echocardiogram showed diffuse hypertrophy, moderately decreased contractility of the left ventricle, and increased intramyocardial signal intensity (Video). The patient was admitted to the hospital for further evaluation. On admission, brain magnetic resonance imaging demonstrated cerebellar atrophy. Cardiac magnetic resonance imaging demonstrated diffuse biventricular hypertrophy and moderately impaired left ventricular systolic function. A wide range of nodular and band-shaped late gadolinium enhancement was detected in the middle and epicardial layers of the left ventricular myocardium. Coronary angiography revealed no significant stenoses of the epicardial coronary arteries.

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Section: Discussionmentioning

confidence: 99%

A Man With Left Ventricular Hypertrophy

2022

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“…Прибор оценивает количество капель и определяет, где реакция прошла, а где -нет. Подразумевается, что в тех микрокаплях, в которые попала целевая ДНК, сигнал будет положительным, а в которые не попала -отрицательным (Hindson et al, 2011;Sofronova et al, 2016). Примеров применения этого подхода в исследованиях по защите растений не так много по причине, как минимум, высокой стоимости анализа.…”

Section: полимеразная цепная реакция (пцр)unclassified

Introduction to Molecular Diagnostics of Insects

et al. 2021

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Insects play an important role in biocenoses due to their abundance and wide (cosmopolitan) distribution. Many insects are crop pests. An effective pest control could be realized in case of proper species identification, which is usually managed by morphological analysis. Molecular methods allow to deep study of many issues of insect biology. In particular, traditional approach can not ordinary identify a species at all stages of their life cycle, whereas molecular methods can it. This review covers a wide range of issues related to the molecular genetic analysis of insects. In the first section we consider the methods of fixation and storage of insect specimens, as well as their impact on DNA quality. Further, we provide general information on population study design. Various schemes of DNA extraction, examples of both express techniques and more thorough protocols for DNA extraction and their purification are provided. In addition, methods of DNA isolation that allow to preserve a specimen integrity for further morphological studies are considered. The methods of DNA quality control are described in detail, that is important for PCR analysis. The last section provides various methods of PCR analysis, that we exemplify by studies aimed to elucidate both fundamental issues and practical problems.

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“…Additionally, the diagnosis of mitochondrial disease remains difficult, as the symptoms are typically non-specific [47]. As the clinical manifestations and severity of mitochondrial-associated diseases are strongly determined by the level of mutant mtDNA, it is a fundamental requirement to develop methods for detecting mutations in heteroplasmic mtDNA with outstanding sensitivity and accuracy, which may contribute to the early diagnosis and dynamic monitoring of mutant mtDNA during disease progression [48].…”

Section: Prenatal Diagnosis Of Genetic Diseasementioning

confidence: 99%

“…According to these data, ddPCR meets the requirements of data reproducibility and high sensitivity in detecting heteroplasmy. In addition, it contributes to the simultaneous detection and quantitative analysis of mtDNA mutations [48].…”

Section: Prenatal Diagnosis Of Genetic Diseasementioning

confidence: 99%

“…In the same year, Rebolledo-Jaramillo et al [50] used ddPCR to validate heteroplasmy and confirmed mutations in mtDNA with reliable outcomes. In 2016, Sofronova et al [48] combined ddPCR with allele-specific probes and specially designed primers to detect Leber's disease-related m.11778G > A mutation and optimized the process with lowered temperatures during the annealing step. According to these data, ddPCR meets the requirements of data reproducibility and high sensitivity in detecting heteroplasmy.…”

Section: Prenatal Diagnosis Of Genetic Diseasementioning

confidence: 99%

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Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Yu¹,

Shen²,

Jiang³

et al. 2017

Cell Physiol Biochem

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Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

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Detection of mutations in mitochondrial DNA by droplet digital PCR

Cited by 9 publications

References 26 publications

A Man With Left Ventricular Hypertrophy

A Man With Left Ventricular Hypertrophy

Introduction to Molecular Diagnostics of Insects

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

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