Automated genomic DNA extraction from saliva using the QIAxtractor
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Abstract:
Venipuncture is an invasive procedure to obtain whole blood in order to obtain high quality and sufficient amounts of genomic DNA. Obtaining DNA from non-invasive sources is preferred by patients, medical doctors and researchers. Saliva collected with cotton swabs (Salivette) is increasingly being used to study chemical compounds, and it can also be a source of DNA. However, extracting DNA from Salivettes is very laborious and time consuming. Therefore, we developed a protocol for automated genomic DNA extraction from saliva collected in Salivette using the QIAxtractor.Saliva (0.1-2.0 mL) was collected by chewing on a Salivette for 1-2 min. A total of 70 samples, collected from healthy volunteers, were extracted with the QIAxtractor robot and a Qiagen DX reagent pack. Quantity and quality was assessed using UV spectrometry and real-time polymerase chain reaction (PCR) (substitution at position -729 in the CYP1A2 gene).The average DNA concentration from the saliva samples was 6.0 microg/mL (95% CI 5.4-6.6 microg/mL). In 100% of the saliva samples, PCR products were detected with an average cycle threshold of 23.1 (95% CI 22.6-23.6).DNA can be extracted in sufficient amounts from Salivette with a fully automated system with a short turnaround time. Real-time PCR can be performed with these samples.Keywords:
genomic DNA
Venipuncture
Buccal swab
The collection of buccal cells with swabs provides a noninvasive method for obtaining genomic DNA for genetic screening. We aimed to study the feasibility of using buccal swabs for genetic screening in patients with exudative age-related macular degeneration (AMD).Blood and buccal swabs were collected for genetic analysis from 65 patients with exudative AMD. Genomic DNA was isolated from either blood or buccal swabs. The yield of genomic DNA from both sources was determined by spectrophotometer. Genotyping for CFH, LOC387715, and HTRA1 Polymorphisms was performed using a method of polymerase chain reaction (PCR) followed by restriction enzyme digestion. Results using genomic DNA from blood or buccal swabs were compared.Three swabs were obtained from each patient, 2 from each side of buccal mucosa, and 1 from both upper and inferior gingival mucosa. From swabs with genomic DNA extracted within a week after sample collection, an average of (3.17 ± 1.46) µg genomic DNA was obtained from swab collected from the left or right side buccal mucosa, and (3.94 ± 1.04) µg from swab collected from the upper and inferior gingival mucosa, with relatively higher yield of genomic DNA from the upper and inferior gingival mucosa (t = 6.79, P < 0.05). From swabs of the left or right side buccal mucosa after being stored at -20°C for 12 months, an average of (3.10 ± 1.17) µg genomic DNA was obtained, which showed no statistically significant difference as compared to the yield of genomic DNA extracted from newly collected swabs (t = 0.59, P > 0.05). In all 65 patients, genomic DNA isolated from either buccal swabs or blood samples showed exactly the same results regarding the genotypes of CFH, LOC387715, and HTRA1 Polymorphisms.Buccal swab is a simple, noninvasive, and reliable source for obtaining genomic DNA. Swabs stored for 12 months at -20°C provide similar amount of genomic DNA as the freshly collected swabs.
Buccal swab
genomic DNA
Buccal mucosa
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genomic DNA
Flora
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genomic DNA
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Objective To optimize bacterial genomic DNA extraction methods of clinical specimens.Methods Different methods of bacterial genomic DNA extraction from pure cultures and clinical specimens were compared.Bacterial DNA Kit,pyrolysis,lysozyme,and the modified method(pyrolysis alkaline lysis combination method) were used.Results The genomic DNA extraction from pure cultures with Bacterial DNA Kit,lysozyme and combination method were be sufficient for PCR amplification(100%).However,the success rate of Gram positive bacteria was 0% with the pyrolysis method.Comparing the Bacterial DNA Kit and the pyrolysis alkaline lysis(NaOH over 20 mmol/L and 2% SDS) for clinical specimens,we found these two both methods were equal for PCR amplification,and concentrations of NaOH and SDS were key factors.Conclusion Pyrolysis alkaline lysis method was suitable for genomic DNA extraction in clinical specimens.
genomic DNA
Alkaline lysis
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[Objective]The research aimed to optimize the extraction methods for genomic DNA in Eomecon chinantha Hance.. [Method]The methods of CTAB and modified CTAB were chosen to extract the genomic DNA in Eomecon chinantha Hance. leaves.[Result]The results showed that the genomic DNA extracted by CTAB method contained many impurities, which were difficult to dissolve and easily degradable.The modified CTAB method was suitable to extract the genomic DNA in Eomecon chinantha Hance..The concentration and extraction rate of DNA from both fresh leaves and dry leaves preserved with silica gel at -80 ℃ were higher. [Conclusion]The study can provide the basis for studying the genetic diversity of Eomecon chinantha Hance..
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[Objective] To explore the extraction methods of genomic DNA of parasitic mites Demodex folliculorum (D.f.) and Demodex brevis (D.b.). [Methods] Mites were disrupted by repeated freezing and thawing in liquid nitrogen. Then,the improved DNA extraction method of mini-insects,alkaline lysis and the commercial extraction kit were used respectively to extract genomic DNA of D.f. and D.b.,which had been kept freezing for ﹤5 or 8-10 months. [Results] The results of protein and nucleic acid radiometer showed the purity and quantity of genomic DNA extracted by the commercial kit were obviously better than those by other methods of mini-insects and alkaline lysis. Clear DNA fingerprints were found in the amplification results of random amplified polymorphic DNA (RAPD). There were obviously different banding patterns between D.f. and D.b. The freezing time affected the quantity of DNA extraction,but had only small effect on the purity and RAPD fingerprints of genomic DNA. The same species of mites had similar banding pattern even using different extraction methods. More and clear bands were detected by the commercial extraction kits and improved DNA extraction method of mini-insects. The bands detected by alkaline lysis method were less and obscure. [Conclusions] The application of repeated freezing-thawing in liquid nitrogen acts effectively to disrupt mites. The mites had better not be kept frozen more than 6 months. The commercial extraction kit is an effective approach to extract genomic DNA of mites. RAPD technique provides a simple method for detecting and classifying D.f. and D.b.
genomic DNA
Primer (cosmetics)
Lysis buffer
Alkaline lysis
DNA profiling
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Seedlings of sixteen mat rush varieties planted in the field,and shoots derived from tissue culture of four varieties were used to investigate the effects of modified CTAB method and Isolation Kit method on the extraction quality of genomic DNA.The results showed that A_260nm and OD values of DNA solution of same material extracted by modified CTAB method were lower than those by isolation kit method.The concentrations of DNA solution were 0.65-4.90 μg/ml with the former method and 5.10-32.80 μg/ml with the latter method.Therefore,it was demonstrated that DNA Isolation Kit method was more suitable for genomic DNA extraction of mat rush.In addition,it was also found that extraction material had the significant impact on the extraction quality of genomic DNA,and the shoots developed by tissue culture were better than seedlings planted in the field.
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Isolation
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Event Abstract Back to Event DNA extraction from Modern Teeth Using Commercially Available DNA Extraction Kits Designed for Buccal Samples Ann-Elodie Robert1*, Caroline Spencer1, Deedra Hughes2, Tracey Dawson-Cruz3 and Murrell Godfrey1 1 University of Mississippi, United States 2 Mississippi Forensic Laboratory, United States 3 Virginia Commonwealth University, United States DNA can be extracted through different media, which includes but is not limited to blood, saliva, bones and even teeth. Most nuclear DNA and mitochondrial DNA (mtDNA) extraction kits are applied to blood or buccal cell samples and many of them are not capable of extracting DNA from ancient remains. The aim of this study was to compare two mtDNA extraction kits used to the extract DNA from teeth. Two extraction kits were examined 1) the QIAmp DNA Blood Mini Kit, used along with a protocol modified by the Dawson-Cruz lab at Virginia Commonwealth University, and 2) a Mitochondrial DNA Polymorphisms in Human Evolution Kit designed by Carolina Biological Supply Company. The first step of this study involved the extraction of DNA from teeth samples. Five unknown male teeth and five unknown female teeth were used in this study. These teeth were extracted within the last 2 years. Once the DNA extraction from the teeth samples were completed, amplification of both the nuclear and mtDNA was performed. Quantitation of the DNA extract was the last part of the experiment to be performed. The successful extraction of both nuclear and mtDNA was initially confirmed through gel electrophoresis and UV-Vis spectroscopy. DNA analysis was then performed using the ABI 7500 Genetic Analyzer to obtain a DNA profile and sex confirmation. Future goals of this study include applying extraction methods on teeth and other remains that range from decades to centuries old. Ultimately the goal is to design a method for extraction and analysis of DNA from ancient remains Keywords: forensic, teeth, DNA, extraction, Analytical, modified kits Conference: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference , Orlando, Florida, United States, 17 Sep - 20 Sep, 2018. Presentation Type: Oral Presentation Topic: Biochemistry Citation: Robert A, Spencer C, Hughes D, Dawson-Cruz T and Godfrey M (2019). DNA extraction from Modern Teeth Using Commercially Available DNA Extraction Kits Designed for Buccal Samples. Front. Chem. Conference Abstract: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference . doi: 10.3389/conf.fchem.2018.01.00020 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Ms. Ann-Elodie Robert, University of Mississippi, Oxford, United States, arobert@go.olemiss.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ann-Elodie Robert Caroline Spencer Deedra Hughes Tracey Dawson-Cruz Murrell Godfrey Google Ann-Elodie Robert Caroline Spencer Deedra Hughes Tracey Dawson-Cruz Murrell Godfrey Google Scholar Ann-Elodie Robert Caroline Spencer Deedra Hughes Tracey Dawson-Cruz Murrell Godfrey PubMed Ann-Elodie Robert Caroline Spencer Deedra Hughes Tracey Dawson-Cruz Murrell Godfrey Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.
Buccal swab
Nuclear DNA
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Genomic DNA from WBCs is widely often used for PCR. Although kits for DNA isolation are in common use, there is scarce information about their performance and about the PCR quality of the genomic DNA obtained. Hence, three different kits, QIAamp blood mini kit, High Pure PCR Template Preparation Kit and the Puregene DNA isolation kit were evaluated on these aspects. Genomic DNA was isolated from whole blood samples with WBC counts ranging from 0.5 to 20*109 WBC/L. The WBC count was used to calculate the amount of genomic DNA. The actual amount of genomic DNA isolated, was determined spectrophotometrically. The DNA extraction efficiency was obtained by dividing the actual amount of DNA by the calculated amount yielded. PCR quality was analysed by measuring Cycle threshold (Ct) values with a quantitative real-time PCR β-globin assay. The extraction efficiency of the three DNA isolation kits was 20% to 40%. Spectrophotometrically determined DNA concentrations correlated inversely with Ct values, regardless of the DNA isolation kit applied, whereas the strongest correlation was obtained for the Puregene DNA isolation kit. WBC counts also correlated inversely with Ct values and here the strongest correlation was found for the QIAamp blood mini kit. In conclusion, the overall performance of the DNA isolation kits was quite comparable (DNA recoveries of 20-40%), albeit the QIAamp blood mini kit yielded the most reproducible extraction efficiencies and lowest Ct values within every WBC count category. Keywords: Cycle theshold (Ct), DNA isolation kit comparison, UV spectrophotometry, real-time PCR, White blood cells (WBC)
genomic DNA
Isolation
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Background: The low yield and quality of buccal-derived genomic DNA have reduced its applicability in various genetic research. The aim of this study was to assess the quantity, purity and genotyping efficiency of genomic DNA isolated from neonatal buccal swabs. Methods: Paired buccal swabs and whole blood samples were collected from 60 neonates with the mean age 5 days (SD=1.57). The genomic DNA quantity and purity were measured by using Infinite® 200 PRO NanoQuant reader and agarose gel electrophoresis. High-resolution melting (HRM) analysis was used to analyse the sequence variants present in uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1 c.211G>A) and nuclear receptor subfamily 1, group I, member 3 (NR1I3 IVS8+116T>G) genes. Results: Buccal swabs provided lower mean genomic DNA concentration (18.78 ± 8.39 ng/µl versus 40.02 ± 13.03 ng/µl), yield (2.63 ± 1.17 µg versus 8.00 ± 2.61 µg). The purity of buccal samples however were inconsistent with 16 samples (26.7%) having A260/280 ratios below 1.8 which indicated protein contamination. Genomic DNA purity for all blood samples were within the ideal range with average absorbance ratios of 1.8−2.0. However, all buccal genomic DNA demonstrated 100% genotype call rates for all variants. A complete genotype concordance was also observed between paired genomic DNA samples. Conclusion: Despite related to a reduced quantity and purity, neonatal buccal genomic DNA could generate reliable HRM genotyping results. Therefore, buccal swab collection is a promising alternative to the invasive blood sampling to provide genomic DNA for genetic analysis involving paediatric population.
Buccal swab
genomic DNA
Agarose gel electrophoresis
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