Allele-Specific Replication Inhibition of Mitochondrial DNA by MITO-PIP Conjugated with Alkylation Reagent

2022 ◽  
pp. 41-65
Author(s):  
Takuya Hidaka
Keyword(s):  
Mitochondrial Dna ◽  
Allele Specific ◽  
Alkylation Reagent

scholarly journals Investigation of Mitochondrial Transfer between Human Erythroblasts

2021 ◽  
Author(s):  
◽  
Brittany Lewer
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Qpcr Assay ◽  
Dna Polymorphisms ◽  
Mitochondrial Transfer ◽  
Hel Cells ◽  
Allele Specific

<p>The increasingly studied phenomenon of mitochondria transferring between cells contrasts the popular belief that mitochondria reside permanently within their cells of origin. Research has identified this process occurring in many tissues such as brain, lung and more recently within the bone marrow. This project aimed to investigate if mitochondria could be transferred between human erythroblasts, a context not previously studied.  Tissue microenvironments can be modelled using co-culture systems. Fluorescence activated cell sorting and a highly sensitive Allele-Specific-Blocker qPCR assay were used to leverage mitochondrial DNA polymorphisms between co-cultured populations. Firstly, HL-60ρ₀ bone marrow cells, without mitochondrial DNA, deprived of essential nutrients pyruvate and uridine were co-cultured in vitro with HEL cells, a human erythroleukemia. Secondly, HEL cells treated with deferoxamine or cisplatin, were cocultured with parental HL-60 cells in vitro. Lastly, ex vivo co-cultures between erythroblasts differentiated from mononuclear cells in peripheral blood were conducted, where one population was treated with deferoxamine.  Co-culture was able to improve recovery when HL-60ρ₀ cells were deprived of pyruvate and uridine. Improved recovery was similarly detected for HEL cells treated with deferoxamine after co-culture with HL-60 cells. Transfer of mitochondrial DNA did not occur at a detectable level in any co-culture condition tested. The high sensitivity of the allele-specific-blocker qPCR assay required completely pure populations to analyse, however this was not achieved using FACS techniques. In conclusion, results have not demonstrated but cannot exclude the possibility that erythroid cells transfer mitochondria to each other.</p>

scholarly journals Direct detection of multiple point mutations in mitochondrial DNA

1997 ◽  
Vol 43 (10) ◽  
pp. 1857-1861 ◽  
Author(s):  
Lee-Jun C Wong ◽  
Dinithi Senadheera
Keyword(s):  
Mitochondrial Dna ◽  
Multiplex Pcr ◽  
Direct Detection ◽  
Point Mutations ◽  
Cost Effective ◽  
Large Deletion ◽  
Multiple Point ◽  
Mtdna Mutations ◽  
Allele Specific ◽  
Mitochondrial Defects

Abstract Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation “hot spots” at tRNAleu(UUR), tRNAlys/ATPase, and ND4 regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.

scholarly journals Investigation of Mitochondrial Transfer between Human Erythroblasts

2021 ◽  
Author(s):  
◽  
Brittany Lewer
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Qpcr Assay ◽  
Dna Polymorphisms ◽  
Mitochondrial Transfer ◽  
Hel Cells ◽  
Allele Specific

<p>The increasingly studied phenomenon of mitochondria transferring between cells contrasts the popular belief that mitochondria reside permanently within their cells of origin. Research has identified this process occurring in many tissues such as brain, lung and more recently within the bone marrow. This project aimed to investigate if mitochondria could be transferred between human erythroblasts, a context not previously studied.  Tissue microenvironments can be modelled using co-culture systems. Fluorescence activated cell sorting and a highly sensitive Allele-Specific-Blocker qPCR assay were used to leverage mitochondrial DNA polymorphisms between co-cultured populations. Firstly, HL-60ρ₀ bone marrow cells, without mitochondrial DNA, deprived of essential nutrients pyruvate and uridine were co-cultured in vitro with HEL cells, a human erythroleukemia. Secondly, HEL cells treated with deferoxamine or cisplatin, were cocultured with parental HL-60 cells in vitro. Lastly, ex vivo co-cultures between erythroblasts differentiated from mononuclear cells in peripheral blood were conducted, where one population was treated with deferoxamine.  Co-culture was able to improve recovery when HL-60ρ₀ cells were deprived of pyruvate and uridine. Improved recovery was similarly detected for HEL cells treated with deferoxamine after co-culture with HL-60 cells. Transfer of mitochondrial DNA did not occur at a detectable level in any co-culture condition tested. The high sensitivity of the allele-specific-blocker qPCR assay required completely pure populations to analyse, however this was not achieved using FACS techniques. In conclusion, results have not demonstrated but cannot exclude the possibility that erythroid cells transfer mitochondria to each other.</p>

scholarly journals High-Sensitivity Detection of the A3243G Mutation of Mitochondrial DNA by a Combination of Allele-Specific PCR and Peptide Nucleic Acid-Directed PCR Clamping

2004 ◽  
Vol 50 (11) ◽  
pp. 2045-2051 ◽  
Author(s):  
Michiyo Urata ◽  
Yui Wada ◽  
Sang Ho Kim ◽  
Worawan Chumpia ◽  
Yuzo Kayamori ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Quantitative Detection ◽  
Wild Type ◽  
Specific Pcr ◽  
Wide Range ◽  
A3243g Mutation ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background: The A3243G mutation of mitochondrial DNA (mtDNA) is involved in many common diseases, including diabetes mellitus and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). For detection of this mutation, allele-specific PCR is highly sensitive but requires strict control of PCR conditions; it thus is not adequate for a routine clinical test. We aimed to develop a routinely available PCR method for quantitative detection of low-level heteroplasmy of the A3243G mutation. Methods: Quantitative allele-specific PCR for the A3243G mutation was performed in the presence of peptide nucleic acid (PNA), in which PNA is complementary to the wild-type mtDNA, with one primer having a 3′ end matched to nucleotide position 3243 of the mutant. Results: With our method, amplification of wild-type mtDNA was suppressed 7000-fold compared with amplification of the mutant mtDNA under a broad range of conditions: DNA, 5–100 ng; annealing temperature, 61–66 °C; and PNA, 1.5–3.5 μmol/L. Hence, 0.1% heteroplasmy of the A3243G mutation can be reliably quantified by this method. Blood samples form 40 healthy volunteers showed &lt;0.06% heteroplasmy, suggesting that 0.1% is diagnostically significant. Conclusions: PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Properties of Isolated Mitochondria of Agaricus Bisporus: Their Relationship to Dormancy and the Germination of Spores

1973 ◽  
Vol 31 ◽  
pp. 458-459
Author(s):  
K. S. McCarty ◽  
R. F. Weave ◽  
L. Kemper ◽  
F. S. Vogel
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Microscopic Examination ◽  
Agaricus Bisporus ◽  
Osmotic Shock ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Isolated Mitochondria ◽  
Dna Strands ◽  
Cytoplasmic Ribosomes

During the prodromal stages of sporulation in the Basidiomycete, Agaricus bisporus, mitochondria accumulate in the basidial cells, zygotes, in the gill tissues prior to entry of these mitochondria, together with two haploid nuclei and cytoplasmic ribosomes, into the exospores. The mitochondria contain prominent loci of DNA [Fig. 1]. A modified Kleinschmidt spread technique1 has been used to evaluate the DNA strands from purified whole mitochondria released by osmotic shock, mitochondrial DNA purified on CsCl gradients [density = 1.698 gms/cc], and DNA purified on ethidium bromide CsCl gradients. The DNA appeared as linear strands up to 25 u in length and circular forms 2.2-5.2 u in circumference. In specimens prepared by osmotic shock, many strands of DNA are apparently attached to membrane fragments [Fig. 2]. When mitochondria were ruptured in hypotonic sucrose and then fixed in glutaraldehyde, the ribosomes were released for electron microscopic examination.

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