Alternative Methods of extracting and Amplifying Dna from lichens

2000 ◽  
Vol 32 (2) ◽  
pp. 189-196 ◽  
Author(s):  
Maria P. Martín ◽  
Katarina Winka
Keyword(s):  
Dna Extraction ◽  
Pcr Amplification ◽  
Alternative Methods ◽  
High Quality

AbstractWe have investigated whether DNA extraction protocols designed specifically for fungi and/or lichens perform better on lichens than do corresponding protocols designed for plants and insects. Two different PCR-amplification protocols were used to evaluate the quality of the DNA extracted with each method. The DNA extractions with highest quality were obtained with the protocols designed for insects and plants, and the most successful amplifications were obtained with Ready-To-Go PCR Beads. This indicates that fungal or lichen specific protocols might not be necessary for successful extraction of high quality DNA from lichens.

scholarly journals Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

Hoehnea ◽  
2019 ◽  
Vol 46 (2) ◽  
Author(s):  
Marília Souza Lucas ◽  
Carolina da Silva Carvalho ◽  
Giovane Böerner Hypolito ◽  
Marina Corrêa Côrtes
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Molecular Techniques ◽  
Tissue Type ◽  
High Quality ◽  
Optimized Protocol ◽  
Palm Species ◽  
Different Tissues

ABSTRACT The application of molecular techniques to tackle ecological and evolutionary questions requires genomic DNA in good quality and quantity. The quality of the isolated DNA, however, can be influenced by the tissue type and the way the sample was conserved and manipulated. Therefore, customizing protocols to improve the DNA isolation and locus amplification is crucial. We optimized a cheap and manual protocol of DNA extraction and microsatellites amplification using five different tissues of a palm species of the brazilian Atlantic Forest. We successfully extracted DNA from all five tissue types. Leaf, stem, and endocarp of non-dispersed seeds presented the highest rates of successful DNA extraction and microsatellite amplification; whereas root, endocarp of dispersed seeds, and embryo showed the lowest quality and quantity. Based on these results, we discussed the implications of using different tissues for studies about seed dispersal, pollination, and population genetics.

scholarly journals Extraction of high-quality genomic DNA and identification of different DNA barcoding markers for chickpea (Cicer arietinum L.)

2020 ◽  
Vol 1 (1) ◽  
pp. 7
Author(s):  
Umavathi Saraswathi ◽  
Lakshmanan Mullainathan
Keyword(s):  
Dna Barcoding ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Genomic Library ◽  
Pcr Amplification ◽  
Slight Modification ◽  
High Quality ◽  
Universal Primer ◽  
Genetic Studies

The genetic studies of individual plants, especially self-pollinated species like chickpea need to be evaluated at the DNA level with the help of molecular markers for identifying genetic variations among the plants. High-quality DNA extraction is a prerequisite for genetic studies. Extraction of intact genomic DNA with high – molecular mass is essential for the study of many molecular biology applications like Polymerase Chain Reaction, endonuclease restriction digestion, southern blot analysis, and also for the construction of a genomic library. Several plant DNA extraction methods are available, even though the DNA isolation methods that give good yield employing both quantity and quality is quite difficult especially for self-pollinated crops like a chickpea. This work was focused on developing a standard protocol for the extraction of genomic DNA and identifying different barcoding markers. The result revealed that the CTAB extraction method with slight modification in protocol had been optimized for DNA isolation. The purified DNA, which was isolated through the CTAB method, had excellent spectral qualities and is efficiently digested by a restriction endonuclease, and is found to be more suitable for long-fragment PCR amplification. DNA barcoding is considered as a promising tool because it provides a practical and standard identification of plants. The isolated DNA sample was processed with a classical DNA barcoding approach by amplifying and sequencing with a universal primer. According to the result, among the different barcoding markers studied, the RbcL and Mat K were found to given the best result for molecular species identification in chickpea.

scholarly journals Saliva leukocytes rather than saliva epithelial cells represent the main source of DNA

2016 ◽  
Vol 24 (1) ◽  
pp. 31-44 ◽  
Author(s):  
Corina Maria Cianga ◽  
Ion Antohe ◽  
Mihaela Zlei ◽  
Daniela Constantinescu ◽  
Petru Cianga
Keyword(s):  
Epithelial Cells ◽  
Dna Extraction ◽  
Peripheral Blood ◽  
Hematological Malignancies ◽  
Mononuclear Cells ◽  
Pcr Amplification ◽  
Hla Typing ◽  
Alternative Methods ◽  
Peripheral Blood Flow ◽  
Immunosuppressed Patients

Abstract Introduction. Several alternative methods to peripheral blood DNA extraction have been implemented so far. Saliva seems to represent a very advantageous type of sample, easy to harvest and able to generate DNA yields comparable to those extracted from blood mononuclear cells. Material and methods. 8 patients suspected of ankylosing spondylitis, 9 patients with various hematological malignancies, displaying post-chemotherapy leucopenia and 30 healthy volunteers were included in our study. DNA was extracted with various commercially available kits and used for HLA typing either by PCR amplification, or by PCR followed by hybridization. Results. Our data regarding HLA typing support already published results regarding the good DNA quality that allows its use in various molecular biology techniques. However, when attempting to use saliva from immunosuppressed patients for DNA extraction we have generated very low yields, comparable again with the ones obtained from peripheral blood. Flow cytometry and immunocytochemistry investigations confirmed the low number of leukocytes present in the saliva of these patients, while the number of epithelial cells was virtually unchanged. Conclusions. The main source of saliva DNA seems to be represented by leukocytes present in this fluid and not by the epithelial cells. Under these circumstances, for immunosuppressed patients saliva cannot represent an alternative to blood when attempting DNA extraction.

scholarly journals Strategy for DNA extraction and detection from insect pests in stored home grain samples RESEARCH

2021 ◽  
Vol 01 (1) ◽  
pp. 24-32
Author(s):  
Ibrahim Abbasi
Keyword(s):  
Dna Extraction ◽  
Insect Pests ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Alternative Methods ◽  
Coi Gene ◽  
Pest Species ◽  
Detection Techniques ◽  
Specificity And Sensitivity ◽  
Washing Method

Stored grains are subjected to infestations with more than 60 species of insects, that responsible for millions of dollars’ loss and cause several health problems including allergies and gastrointestinal disorders. Traditional detection techniques are laborious, expensive and not sensitive to detect insect contamination at the egg and larvae stages. Therefore, alternative methods are needed for rapid and sensitive detection. One obvious approach is to develop a molecular approach utilizing genetic information of the potential insect species that infest grains for amplification of specific target gene fragment utilizing polymerase chain reaction [PCR]. In the present study, a number of known infested grain samples were used in standardizing a method to isolate larvae and adult insects that were based on centrifugation washing method and a filtration washing method. The isolated insects were subjected to DNA extraction and PCR amplification of defined regions of cytochrome oxidase I (COI) gene followed by sequencing to identify the di"erent pest species. For PCR amplification new primers were designed and for this purpose the obtained COI sequences from di"erent insects were aligned to design two sets of primers (named: COI-PCR4 and COI-PCR5) specific for the indicated insect mitochondrial COI gene. The designed primers were tested for their specificity and sensitivity. The suitability of PCR primers and DNA extraction methods were evaluated on eleven samples of commercial grains utilizing each primer set with the two extraction methods.

scholarly journals DNA Extraction from Several Cacti

HortScience ◽  
2000 ◽  
Vol 35 (6) ◽  
pp. 1124-1126 ◽  
Author(s):  
Candelario Mondragon-Jacobo ◽  
Natalia Doudareva ◽  
Bruce P. Bordelon
Keyword(s):  
Dna Extraction ◽  
Pcr Amplification ◽  
Hexadecyltrimethylammonium Bromide ◽  
Digestion Time ◽  
Chain Reaction ◽  
High Quality ◽  
Banding Patterns ◽  
Extraction Buffer ◽  
Ctab Method ◽  
Polymerase Chain

A method for extraction of high quality DNA from four Opuntia sp. and other cacti using a hexadecyltrimethylammonium bromide (CTAB) method is described. These plants typically contain high levels of mucilages, complex polysaccharide compounds that bind water, thus preventing DNA extraction by common miniprep methods. The method involves adjusting the amount of tissue used according to species and age, followed by processing in an extraction buffer to separate coarse material. Extended centrifugation and digestion time in a separation buffer with CTAB (2%) was used. Exposing tissue to both buffers maintained polysaccharides in solution and allowed easier recovery of the aqueous phase that contains the DNA. We found that 5-8 g were needed to obtain up to 153 μg·g-1 of DNA from tender tissue. Old tissue yielded 26% less. Extraction of DNA from 5-g samples of tender tissue of the ornamental cacti Stenocereus sp., Cleistocactus sp., and Echinocereus sp. was successful. For these species, average yields ranged from 25 to 53 μg per sample. The DNA obtained was suitable for polymerase chain reaction (PCR) amplification, producing clear, distinctive, and reproducible banding patterns useful for a variety of applications.

scholarly journals High Quality of Bacterial DNA Extraction from Corbicula fluminea Tissue in Kelantan

2020 ◽  
Vol 16 (2) ◽  
pp. 161-165
Author(s):  
Koh Han Dee ◽  
Suganthi Appalasamy ◽  
Siti Nor Aini Md Nasir ◽  
Faizuan Abdullah ◽  
Maryana Mohamad Nor ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Cetyltrimethylammonium Bromide ◽  
Corbicula Fluminea ◽  
High Quality ◽  
Bacterial Dna ◽  
Tissue Surface ◽  
High Integrity ◽  
Effectiveness And Efficiency ◽  
Bacterial Dna Extraction

Corbicula fluminea served as a popular traditional food in Kelantan, Malaysia. This clam is highly consumed by the local people from the last decade. However, there is lack of the study that obtain high quality of bacterial metagenome deoxyribonucleic acid (DNA) extraction from C. fluminea. Improper DNA extraction protocol always fails to extract a high integrity of bacterial DNA bands. This study compares the effectiveness and efficiency of conventional cetyltrimethylammonium bromide (CTAB) protocol, a commercial kit and modified CTAB protocol for bacterial DNA extraction from the soft tissue surface of raw C. fluminea. The instruments used to examine the quality of the extracted bacterial DNA were DeNovix DS-11 spectrophotometer, gel electrophoresis machine and UV transilluminator. The results showed that the bacterial DNA extracted from modified CTAB protocol the highest purity and integrity with the A₂₆₀/A₂₈₀ ratio of 1.92 ± 0.01 as well as the DNA band with minimum smear. This conclude that modified CTAB protocol is the best among the three extraction protocols for the bacterial extraction from the C. fluminea.

scholarly journals Constructing high-quality rest facilities to maximise performance and ensure patient safety

2021 ◽  
Vol 10 (3) ◽  
pp. e001403
Author(s):  
Geeth Silva ◽  
Aiken Yam ◽  
Jessica Court ◽  
Rabia Imtiaz ◽  
Cath Chisholm
Keyword(s):  
Patient Safety ◽  
Well Being ◽  
Recovery Phase ◽  
Alternative Methods ◽  
Junior Doctors ◽  
High Quality ◽  
Ensure Patient Safety ◽  
Being There ◽  
Exception Reporting

IntroductionJunior doctors are working in an increasingly overstretched National Health Service. In 2018, Kettering General Hospital (KGH) was awarded £60 800 of government funds to create high-quality rest facilities and improve junior doctor well-being.MethodsAn audit and survey in KGH identified the structural and functional improvements needed. From November 2019 to June 2020, £47 841.24 was spent on creating new rest facilities. On completion, a postaction review assessed how the changes impacted morale, well-being and quality of patient care.ResultsThe majority of doctors were happy with the new rest areas (60%), a majority felt that they would use the on-call room area (63%) and the renovation improved morale and well-being. There was an increased ability to take breaks. However, the majority of doctors are not exception-reporting missing breaks: 79% (2019), 74% (2020).Conclusions and ImplicationsThis report recommends the maintenance of increased staffing levels and rest facilities during the recovery phase of COVID-19. The remaining £12 958.76 should be directed at sustaining the quality of KGH rest facilities. Lastly, the rate of exception-reporting must be increased through improving awareness, exploring alternative methods and supporting the action when necessary. The continual investment into rest facilities ensures workforce well-being and translates into patient safety.

scholarly journals Vouchering of Forensically Important Fly Specimens by Nondestructive DNA Extraction

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Seong Yoon Kim ◽  
Seong Hwan Park ◽  
Huguo Piao ◽  
Ukhee Chung ◽  
Kwang Soo Ko ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Pcr Amplification ◽  
Nondestructive Method ◽  
Anatomical Structure ◽  
Whole Body ◽  
Anatomical Structures ◽  
Taxonomic Importance ◽  
Voucher Specimens ◽  
Phaenicia Sericata

DNA extraction frequently requires destruction of whole samples. However, when the sample is very rare or has taxonomic importance, nondestructive DNA extraction is required for preservation of voucher specimens. In the case of arthropod specimens, minor anatomical structures such as a single leg or a single wing are often sacrificed instead of the whole body for DNA extraction. In an attempt to save the entire anatomical structure of specimens, several authors tried to brew the whole specimen in a lysis buffer and to extract DNA from the “soup.” We applied this nondestructive DNA extraction technique to a forensically important blowfly species, Phaenicia sericata. With nondestructive DNA extraction, a satisfactory quantity and quality of DNA for PCR amplification was obtained with only minimal anatomical disruptions that do not alter the morphologic identification. This nondestructive method may be applicable to DNA extraction of rare samples as well as vouchering of regular fly samples.

scholarly journals Comparison and Validation of Ichthyoplankton DNA Extraction Methods

2021 ◽  
Vol 4 (4) ◽  
pp. 87
Author(s):  
Diouri Lamia ◽  
Uwiringiyeyezu Théophile ◽  
Abdelouahab Hinde ◽  
Malki Mohamed ◽  
Baibai Tarik ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Classical Method ◽  
Pcr Amplification ◽  
Temporal Distribution ◽  
Extraction Methods ◽  
Direct Pcr ◽  
Fisheries Resources ◽  
Morphological Criteria

Ichthyoplankton is the cluster of planktonic organisms that consists of fish eggs and larvae. These planktonic stages belong to the temporary zooplankton, representing future exploitable stocks. The study of the early ontogenesis of fish plays a key role in the understanding and evaluation of these populations through the study of their abundance and their spatio-temporal distribution. To better understand and protect these fisheries resources, it is essential to identify the different stages of fish embryonic development. This identification is usually performed using the classical method, based on morphological criteria under a binocular magnifying glass; however, this methodology is not always sufficient and is time consuming and, therefore, it is necessary to rely increasingly on molecular tools. The major problem with these tools is the yield and quality of the nucleic acids extracted from ichthyoplankton, especially in the case of eggs, which are small. Several methods have been used for DNA extraction from ichthyoplankton, either automated or manual, but very often from larvae or adults. In the present work, five fish egg DNA extraction protocols were compared based on their DNA yield and extraction quality, verified by agarose gel electrophoresis and quantitative PCR amplification. The results showed that extraction by our heat-protocol for direct PCR (Hp-dPCR) presents the simplest and cheapest protocol of all the kits used in this study, providing a sufficient quantity and quality of nucleic acids to be used for PCR amplification, and being within the reach of third world laboratories that often do not have sufficiently large budgets to obtain automated kits.

Influence of technology parameters of free-cutting steel continuous casting on the billet internal structure

2020 ◽  
Vol 76 (2) ◽  
pp. 139-142
Author(s):  
A. T. Kunakbaeva ◽  
A. M. Stolyarov ◽  
M. V. Potapova
Keyword(s):  
Regression Analysis ◽  
Continuous Casting ◽  
Internal Structure ◽  
Sulfur Content ◽  
Manganese Content ◽  
High Quality ◽  
Correlation And Regression Analysis ◽  
Cutting Steel ◽  
Continuously Cast

Free-cutting steel gains specific working properties thanks to the high content of sulfur and phosphorus. These elements, especially sulfur, have a rather high tendency to segregation. Therefore, segregation defects in free-cutting steel continuously cast billets can be significantly developed. The aim of the work was to study the influence of the chemical composition of freecutting steel and casting technological parameters on the quality of the macrostructure of continuously cast billets. A metallographic assessment of the internal structure of cast metal made of free-cutting steel and data processing by application of correlation and regression analysis were the research methods. The array of production data of 43 heats of free-cutting steel of grade A12 was studied. Steel casting on a five-strand radial type continuous casting machine was carried out by various methods of metal pouring from tundish into the molds. Metal of 19 heats was poured with an open stream, and 24 heats – by a closed stream through submerged nozzles with a vertical hole. High-quality billets had a cross-sectional size of 150×150 mm. The macrostructure of high-quality square billets made of free-cutting steel of A12 grade is characterized by the presence of central porosity, axial segregation and peripheral point contamination, the degree of development of which was in the range from 1.5 to 2.0 points, segregation cracks and strips – about 1.0 points. In the course of casting with an open stream, almost all of these defects are more developed comparing with the casting by a closed stream. As a result of correlation and regression analysis, linear dependences of the development degree of segregation cracks and strips both axial and angular on the sulfur content in steel and on the ratio of manganese content to sulfur content were established. The degree of these defects development increases with growing of sulfur content in steel of A12 grade. These defects had especially strong development when sulfur content in steel was of more than 0.10%. To improve the quality of cast metal, it is necessary to have the ratio of the manganese content to the sulfur content in the metal more than eight.

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