ResearchPad - dna-separation https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays]]> https://www.researchpad.co/article/elastic_article_14590 Highly sensitive detection of HIV-1 nucleic acids is of critical importance for evaluating treatment interventions superimposed on combination antiretroviral therapy (cART) in HIV-1 infected individuals. SIV infection of rhesus macaques models many key aspects of human HIV-1 infection and plays a key role in evaluation of approaches for prevention, treatment and attempted eradication of HIV infection. Here we describe two droplet digital PCR (ddPCR) assays, a ddPCR DNA assay and an RT-ddPCR RNA assay for detecting simian immunodeficiency virus (SIV) on the RainDance platform. We demonstrate that RainDance ddPCR can tolerate significantly higher cell DNA input without inhibition on a per reaction basis (compared to both qPCR and Bio-Rad ddPCR), thus allowing a large quantity of sample to be analyzed in each reaction. In addition, the combination of a high processivity RT enzyme and RainDance ddPCR could overcome inhibition in severely inhibited (99.99% inhibition in qPCR quantification) viral RNA samples. These assays offer valuable tools for assessing low level viral production/replication and strategies for targeting residual virus in the setting of cART suppression of viral replication. The methodologies presented here can be adapted for a broad range of applications where highly sensitive nucleic acid detection is required.

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<![CDATA[A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR]]> https://www.researchpad.co/article/5989d9f8ab0ee8fa60b70e34

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as “rain” in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

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<![CDATA[Unexpected DNA Loss Mediated by the DNA Binding Activity of Ribonuclease A]]> https://www.researchpad.co/article/5989d9e4ab0ee8fa60b6a8b3

Ribonuclease A (RNase A) is widely used in molecular biology research both for analytical assays and for nucleic acid preparation. The catalytic mechanism of RNase A is well understood and absolutely precludes activity on DNA; however anecdotal reports of DNA degradation by RNase A are not uncommon. Here we describe a mechanism by which RNase A treatment can lead to apparent DNA degradation. This results from the surprising finding that RNase A remains functional in a phenol:chloroform mixture, to our knowledge the only enzyme that survives this highly denaturing solvent environment. Although RNase A does not cleave the DNA backbone it is capable of binding to DNA, forming stable RNase A-DNA complexes that partition to the interphase or organic phase during phenol:chloroform purification. The unexpected survival of the RNase A DNA-binding activity in phenol means that these complexes are not dissolved and a substantial amount of RNase A-bound DNA is permanently removed from the aqueous phase and lost on phase separation. This effect will impact DNA recovery from multiple procedures and is likely to represent a source of sequence bias in genome-wide studies. Our results also indicate that the results of analytical studies performed using RNase A must be considered with care.

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<![CDATA[Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments]]> https://www.researchpad.co/article/5989db11ab0ee8fa60bcc1e6

In this study, GelGreen was investigated as a replacement for SYBR® Safe to stain DNA in cesium chloride (CsCl) density gradients for DNA-stable isotope probing (SIP) experiments. Using environmental DNA, the usage of GelGreen was optimized for sensitivity compared to SYBR® Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen was more sensitive than SYBR® Safe, while the optimal dosage (15X concentration) needed was approximately one-third of SYBR® Safe, suggesting that its sensitivity was three times more superior than SYBR® Safe. At these optimal parameters, the detection limit of GelGreen-stained environmental DNA was as low as 0.2 μg, but the usage of 0.5 μg environmental DNA was recommended to produce a more consistent DNA band. In addition, a modified needle extraction procedure was developed to withdraw DNA effectively by fractionating CsCl density gradients into four or five fractions. The successful application of GelGreen staining with 13C-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR® Safe in CsCl density gradients for DNA-SIP assays.

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