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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes coronavirus disease 2019 (COVID-19) and has affected over 25 million people worldwide as of ...August 31, 2020. To aid in the development of diagnostic kits for rapid and sensitive detection of the virus, we evaluated a combination of polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques. Here, we compared conventional PCR and loop-mediated isothermal amplification (LAMP) methods with hybrid techniques such as polymerase chain displacement reaction (PCDR) and a newly developed PCR-LAMP method. We found that the hybrid methods demonstrated higher sensitivity and assay reaction rates than those of the classic LAMP and PCR techniques and can be used to for SARS-CoV-2 detection. The proposed methods based on the modern hybrid amplification techniques markedly improve virus detection and, therefore, can be extremely useful in the development of new diagnostic kits.
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
and
DNA polymerases to a novel
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.
The methods of forming the components of fuzzy knowledge base in the form of basic digital plan-scheme of territories determined by the morphology of satellite images, natural data and the results of ...subjective assessments are considered. Variants of implementation of the algorithm of identification of the increased reliability taking into account fuzziness of initial data are investigated. During the development of the model of modern monitoring of territories in the interests of economic and social development of the population, it became obvious that the characteristics of the information with which this model should operate, does not correspond to those objects that describe the "deterministic" mathematics, that is, having certainty, accuracy, completeness, isolation, consistency, etc. Man-machine systems (namely, these should include the system of space monitoring) and components of the apparatus of representation of knowledge in them, in reality, reflect those properties of the human model of the world that do not fit into the deterministic mathematics and are characterized by the presence of "non-factors" - incompleteness, lack of accuracy, nonclosure, possibility of contradictions, etc. In this paper we propose a developed and successfully applied in practice by the authors’ method of accounting for non-factors in the identification of objects of the Earth's surface. Within the framework of this method, the following sequence of actions is performed: a vector three-dimensional model of the reference object is obtained by geometric construction, then, changing its position in space (rotation, reflection, scaling), a number of the above parameters are obtained, which are stored and used in the future for recognition to recreate the corresponding angle of the reference object. The paper considers the importance of non-factors in the process of identification of objects of the earth's surface during monitoring using satellite data, their condition, the variant to overcome the negative impact of different types of fuzzy initial monitoring data and improve the reliability of its results on the principles of quorum reservation is proposed.
The site‐specific endonuclease Bme216I was isolated as a homogeneous preparation by chromatography on phosphocellulose, hydroxyapatite and heparin‐agarose. The molecular mass of the enzyme, ...determined by gel filtration and by electrophoresis under denaturing conditions, was found to be 60 kDa and 30 kDa respectively. These data indicate that the native enzyme consists of two identical subunits. The enzyme recognized the pentanucleotide sequence and cleaves the sequence as indicated by arrows. The optimal concentration for endonuclease reaction is 6–7 mM Mg2+. The endonuclease relaxes its specificity in the presence of glycerol or dimethyl sulfoxide at low Mg2+ concentration (1–3 mM). Methylase Bme216I, which protects DNA against endonuclease Bme216I action by DNA methylation, was isolated from the same bacterial strain.
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