scholarly journals A reassessment of several erstwhile methods for isolating DNA fragments from agarose gels

3 Biotech ◽  
2021 ◽  
Vol 11 (3) ◽  
Author(s):  
Xia Gao ◽  
Keyin Zhang ◽  
Tianzhu Lu ◽  
Yan Zhao ◽  
Haiyan Zhou ◽  
...  
Keyword(s):  
Filter Paper ◽  
Dna Fragments ◽  
Dialysis Tubing ◽  
Nested Polymerase Chain Reaction ◽  
Agarose Gels ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Dna Precipitation ◽  
Commercial Kits ◽  
Biology Research

AbstractMolecular biology research often requires extraction of DNA fragments from agarose gels. In the past decades, there have been many methods developed for this purpose. Currently most researchers, especially novices, use commercial kits for this extraction, although these kits cost money and the procedures involved are not necessarily easier than some erstwhile methods. We herein reintroduce and reassess several simple and cost-free older methods. One method involves excising a slice of the gel containing the DNA fragment, followed by a thaw-and-freeze procedure to release the DNA from the gel slice into the gel-making buffer. The second method involves a dialysis tubing and requires electroelution of the DNA from the gel slice in the tubing. The third one is to centrifuge the gel slice to release the DNA. The fourth method requires electro-transfer of the DNA from the gel into a filter paper, while the fifth one includes either allowing the DNA in the slice to be dissolved into a buffer or dissolving the DNA-containing gel slice, followed by DNA precipitation with ethanol or isopropanol. The strengths and weaknesses of these methods are discussed to assist researchers in making their choice. We also point out that some of the end uses of the DNA fragment in the agarose gel may not actually require extraction of the DNA. For instance, a tiny DNA-containing gel block or filter paper can be directly used as the template in a nested or semi-nested polymerase chain reaction to preliminarily determine the identity of the DNA fragment.

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction

2020 ◽  
Vol 58 (4) ◽  
pp. 527-532 ◽  
Author(s):  
Jee-Soo Lee ◽  
Miyoung Kim ◽  
Moon-Woo Seong ◽  
Han-Sung Kim ◽  
Young Kyung Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Analytical Sensitivity ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Specimen Type ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Ctdna Analysis ◽  
Tumor Dna

AbstractBackgroundChoosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences.MethodsWe isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR.ResultsThe amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts.ConclusionsOur results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

scholarly journals MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors

2020 ◽  
Vol 36 (11) ◽  
pp. 3322-3326
Author(s):  
Michael Schwarz ◽  
Marius Welzel ◽  
Tolganay Kabdullayeva ◽  
Anke Becker ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Web Application ◽  
De Novo ◽  
Supplementary Information ◽  
Dna Fragments ◽  
Synthetic Dna ◽  
Custom Made ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Error Probabilities

Abstract Summary The development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes. Availability and implementation MESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

scholarly journals Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

2017 ◽  
Vol 69 (6) ◽  
pp. 1443-1450 ◽  
Author(s):  
M.P. Campos ◽  
M.F. Madeira ◽  
D.A. Silva ◽  
M.S. Solcà ◽  
O.M. Espíndola ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Healthy Skin ◽  
Sample Collection ◽  
Chain Reaction ◽  
Dna Recovery ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Polymerase Chain ◽  
Commercial Kits

ABSTRACT The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery

Detection of hepatitis “C” virus in formalin-fixed liver tissue by nested polymerase chain reaction

1992 ◽  
Vol 37 (4) ◽  
pp. 310-314 ◽  
Author(s):  
Richard Sallie ◽  
Anne Rayner ◽  
Bernard Portmann ◽  
A. L. W. F. Eddleston ◽  
Roger Williams
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Liver Tissue ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Formalin Fixed

scholarly journals A PCR-based Assay for Distinguishing between A1 and A2 Mating Types of Phytophthora capsici

2017 ◽  
Vol 142 (4) ◽  
pp. 260-264
Author(s):  
Ping Li ◽  
Dong Liu ◽  
Min Guo ◽  
Yuemin Pan ◽  
Fangxin Chen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Mating Type ◽  
Phytophthora Capsici ◽  
Mating Types ◽  
Sequence Repeat ◽  
Chain Reaction ◽  
Plant Parasite ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Microsatellite Diversity

Sexual reproduction in the plant parasite Phytophthora capsici Leonian requires the interaction of two distinct mating types, A1 and A2. Co-occurrence of these mating types can enhance the genetic diversity of P. capsici and alter its virulence or resistance characteristics. Using an intersimple sequence repeat (ISSR) screen of microsatellite diversity, we identified, cloned, and sequenced a novel 1121-base pair (bp) fragment specific to the A1 mating type of P. capsici. Primers Pcap-1 and Pcap-2 were designed from this DNA fragment to specifically detect the A1 mating type. Polymerase chain reaction (PCR) using these primers amplified an expected 997-bp fragment from known A1 mating types, but yielded a 508-bp fragment from known A2 mating types. This PCR-based assay could be adapted to accurately and rapidly detect the co-occurrence of A1 and A2 P. capsici mating types from field material.

The fate of a genetically modifiedPseudomonasstrain and its transgene during the composting of poultry manure

2004 ◽  
Vol 50 (6) ◽  
pp. 415-421 ◽  
Author(s):  
J Guan ◽  
J L Spencer ◽  
M Sampath ◽  
J Devenish
Keyword(s):  
Polymerase Chain Reaction ◽  
Incubation Temperature ◽  
Genetically Modified ◽  
Chicken Manure ◽  
Detection Sensitivity ◽  
Plate Count ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Soil Microcosms ◽  
Polymerase Chain

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 × 1010CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 °C or above for one or more days. In contrast, in the compost microcosms incubated at 23 °C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 °C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.Key words: composting, genetically modified Pseudomonas strain, transgene, polymerase chain reaction.

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