| 1. |
- Blagodatskikh, Konstantin A., et al.
(författare)
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Improved DOP-PCR (iDOP-PCR) : A robust and simple WGA method for efficient amplification of low copy number genomic DNA
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:9
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Tidskriftsartikel (refereegranskat)abstract
- Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high-and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.
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| 2. |
- Ignatov, Konstantin B., et al.
(författare)
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A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification
- 2014
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 57:2, s. 81-87
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Tidskriftsartikel (refereegranskat)abstract
- The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Tag DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.
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| 3. |
- Ignatov, Konstantin B., et al.
(författare)
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Fragmentation Through Polymerization (FTP) : A new method to fragment DNA for next-generation sequencing
- 2019
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14:4
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Tidskriftsartikel (refereegranskat)abstract
- Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends.
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| 4. |
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| 5. |
- Kramarova, Tatiana V., et al.
(författare)
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Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-Fo subunit P1 isoform
- 2008
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 22:1, s. 55-63
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Tidskriftsartikel (refereegranskat)abstract
- Despite the significance of mitochondrial ATP synthase for mammalian metabolism, the regulation of the amount of ATP synthase in mammalian systems is not understood. As brown adipose tissue mitochondria contain very low amounts of ATP synthase, relative to respiratory chain components, they constitute a physiological system that allows for examination of the control of ATP synthase assembly. To examine the role of the expression of the P1-isoform of the c-F-o subunit in the biogenesis of ATP synthase, we made transgenic mice that express the P1-c subunit isoform under the promoter of the brown adipose tissue-specific protein UCP1. In the resulting UCP1p1 transgenic mice, total P1-c subunit mRNA levels were increased; mRNA levels of other F1F(o)-ATPase subunits were unchanged. In isolated brown-fat mitochondria, protein levels of the total c-Fo subunit were increased. Remarkably, protein levels of ATP synthase subunits that are part of the F-1-ATPase complex were also increased, as was the entire Complex V. Increased ATPase and ATP synthase activities demonstrated an increased functional activity of the F1Fo-ATPase. Thus, the levels of the c-F-o subunit P1-isoform are crucial for defining the final content of the ATP synthase in brown adipose tissue. The level of c-F-o subunit may be a determining factor for F1Fo-ATPase assembly in all higher eukaryotes.-Kramarova, T. V., Shabalina, I. G., Andersson, U., Westerberg, R., Carlberg, I., Houstek, J., Nedergaard, J., Cannon, B. Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-F-o subunit P1 isoform.
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| 6. |
- Shabalina, Irina G, et al.
(författare)
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Cold tolerance of UCP1-ablated mice : A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal, fatty acid- or ROS-induced uncoupling or enhanced GDP effects.
- 2010
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2728. ; 1797:6-7, s. 968-80
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Tidskriftsartikel (refereegranskat)abstract
- Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4 degrees C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(-/-) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(-/-) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(-/-) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(-/-), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.
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| 7. |
- Shabalina, Irina G., et al.
(författare)
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The Environmental Pollutants Perfluorooctane Sulfonate and Perfluorooctanoic Acid Upregulate Uncoupling Protein 1 (UCP1) in Brown-Fat Mitochondria Through a UCP1-Dependent Reduction in Food Intake
- 2015
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Ingår i: Toxicological Sciences. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 146:2, s. 334-343
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Tidskriftsartikel (refereegranskat)abstract
- The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice. Correspondingly, PFOA/PFOS treatment induced recruitment of brown adipose tissue mitochondria: increased oxidative capacity and increased UCP1-mediated oxygen consumption (thermogenesis). In mice pair-fed to the food intake during PFOA/PFOS treatment in wildtype mice, brown-fat mitochondrial recruitment was also induced. We conclude that we have uncovered the existence of a regulatory component of food intake that is dependent upon brown adipose tissue thermogenic activity. The possible environmental consequences of this novel PFOA/PFOS effect (a possible decreased fitness) are noted, as well as the perspectives of this finding on the general understanding of control of food intake control and its possible extension to combatting obesity.
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