Optimizing DNA Yield for Molecular Analysis from Cytologic Preparations: Extraction Method and Glass Slide Type

A highly efficient strategy for recovery of genomic DNA from bone and tooth samples is presented by complete demineralization of the bone pieces or intact tooth with high concentration of EDTA followed by spin column treatment without the need of mechanical grinding or cryogenic method for pulverizing the samples. The DNA yield was between 8 and 12 ng/?l from approximately 1 ?2 g of the starting material. Completed DNA profiles were obtained from of all the bones (52) and tooth (270) samples received from the unidentified victims from a recent building collapse, the Rana Plaza disaster in Dhaka, Bangladesh. DOI: http://dx.doi.org/10.3329/dujbs.v23i2.20089 Dhaka Univ. J. Biol. Sci. 23(2): 101-107, 2014

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Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis

Acta Cytologica ◽

10.1159/000519336 ◽

2021 ◽

pp. 1-7

Author(s):

Shruti Gupta ◽

Upasana Gautam ◽

Shaily Susheilia ◽

Baneet Bansal ◽

Radha Uppal ◽

...

Keyword(s):

Sodium Alginate ◽

Molecular Analysis ◽

Low Cost ◽

Nuclear Marker ◽

Fine Needle Aspirates ◽

Dna Yield ◽

Morphological Evaluation ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Background: Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation – plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. Design: A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ2 test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. Results: CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). Conclusion: SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

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A modified SDS-based DNA extraction method from raw soybean

Bioscience Reports ◽

10.1042/bsr20182271 ◽

2019 ◽

Vol 39 (2) ◽

Cited By ~ 10

Author(s):

Yimiao Xia ◽

Fusheng Chen ◽

Yan Du ◽

Chen Liu ◽

Guanhao Bu ◽

...

Keyword(s):

Dna Extraction ◽

Extraction Method ◽

Pcr Amplification ◽

Isoamyl Alcohol ◽

Monitoring Methods ◽

Detection Techniques ◽

Dna Yield ◽

Seed Flour ◽

Dna Extraction Method ◽

Lysis Buffer

Abstract Soybean is the most important genetically modified (GM) oilseed worldwide. Regulations relating to the approval of biotech soybean varieties and product labeling demand accurate and reliable detection techniques to screen for GM soya. High-quality extracted DNA is essential for DNA-based monitoring methods. Thus, four widely used protocols (SDS, CTAB, DP305, and DNeasy Plant Mini Kit) were compared in the present study to explore the most efficient DNA extraction method for raw soya matrix. The SDS-based method showed the highest applicability. Then crucial factors influencing DNA yield and purity, such as SDS lysis buffer component concentrations and organic compounds used to isolate DNA, were further investigated to improve the DNA obtained from raw soybean seeds, which accounts for the innovation of this work. As a result, lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and organic reagents including chloroform/isoamyl alcohol (24:1, v/v) (C: I), isopropanol, and ethanol corresponding to the extraction and first and second precipitation procedures, respectively, were used in the optimized SDS method. The optimized method was verified by extracting approximately 2020–2444 ng DNA/mg soybean with A260/280 ratios of 1.862–1.954 from five biotech and non-biotech soybean varieties. Only 0.5 mg of soya was required to obtain enough DNA for PCR amplification using the optimized SDS-based method. These results indicate that the screening protocol in the present study achieves the highest suitability and efficiency for DNA isolation from raw soya seed flour.

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A novel method for liquid-phase extraction of cell-free DNA for detection of circulating tumor DNA

Scientific Reports ◽

10.1038/s41598-021-98815-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Filip Janku ◽

Helen J. Huang ◽

David Y. Pereira ◽

Masae Kobayashi ◽

Chung Hei Chiu ◽

...

Keyword(s):

Liquid Phase ◽

Solid Phase Extraction ◽

Extraction Method ◽

Solid Phase ◽

Cancer Diagnostics ◽

Phase Extraction ◽

Plasma Samples ◽

Cell Free Dna ◽

Dna Yield ◽

Free Dna

AbstractLow yields of extracted cell-free DNA (cfDNA) from plasma limit continued development of liquid biopsy in cancer, especially in early-stage cancer diagnostics and cancer screening applications. We investigate a novel liquid-phase-based DNA isolation method that utilizes aqueous two-phase systems to purify and concentrate circulating cfDNA. The PHASIFY MAX and PHASIFY ENRICH kits were compared to a commonly employed solid-phase extraction method on their ability to extract cfDNA from a set of 91 frozen plasma samples from cancer patients. Droplet digital PCR (ddPCR) was used as the downstream diagnostic to detect mutant copies. Compared to the QIAamp Circulating Nucleic Acid (QCNA) kit, the PHASIFY MAX method demonstrated 60% increase in DNA yield and 171% increase in mutant copy recovery, and the PHASIFY ENRICH kit demonstrated a 35% decrease in DNA yield with a 153% increase in mutant copy recovery. A follow-up study with PHASIFY ENRICH resulted in the positive conversion of 9 out of 47 plasma samples previously determined negative with QCNA extraction (all with known positive tissue genotyping). Our results indicate that this novel extraction technique offers higher cfDNA recovery resulting in better sensitivity for detection of cfDNA mutations compared to a commonly used solid-phase extraction method.

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MagnaExtract, a novel magnetic bead-based extraction method for the molecular detection of antimicrobial resistance genes in fresh water

10.1101/2021.04.23.439981 ◽

2021 ◽

Author(s):

Rachel L. Byrne ◽

Derek Cocker ◽

Ghaith Alyayyoussi ◽

Madalitso Mphasa ◽

Mary Charles ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Dna Extraction ◽

Resistance Genes ◽

Water Samples ◽

Extraction Method ◽

Bacterial Isolate ◽

Magnetic Bead ◽

Dna Yield ◽

Dna Extraction Method ◽

Antimicrobial Resistance Genes

ABSTRACTBackgroundEnvironmental water samples are increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and next generation sequencing (NGS) offer a potentially inclusive surveillance platform for a wide range of AMR genes. However, molecular methods are dependent upon the extraction of DNA of high yield and quality. Current options for DNA extraction from complex environmental matrices for downstream molecular applications are either expensive or low yielding. We present here a novel magnetic bead-based DNA extraction method, for the detection of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract.MethodsMagnaExtract involves initial filtration of 250ml freshwater, followed by an overnight incubation of the filter in 15ml buffered peptone water (BPW), common procedure in microbiology laboratories. 200µl is then taken for a boil (95°C) and spin step and mixed with magnetic beads to bind DNA. Following washes with ethanol, the DNA is eluted in nuclease-free water. To determine the effectiveness of this method, 98 freshwater samples were collected from two rivers in Southern Malawi, and DNA was isolated using the MagnaExtract method, two commercial Qiagen (Germany) kits; PowerWater and DNeasy Blood and tissue, alongside a boil and spin of BPW, and a boil and spin from bacterial isolate grown on agar media. All samples were screened with a high-resolution melt (HRM) PCR panel previously validated for the detection of third generation cephalosporin and carbapenem ARGs. We compared the DNA yield obtained using all extraction methods, as well as the identification of each ARG.ResultsDNA yield using MagnaExtract was statistically greater than both boil and spin methods and DNeasy Blood & Tissue (Qiagen, Germany). DNA yield was slightly lower than using PowerWater (Qiagen) but the difference was not statistically significant. MagnaExtract was the only method to identify ARGs in all 98 water samples compared with PowerWater (n=82), DNeasy (n=95) boilate of BPW (n=75) and boilate of bacterial isolate (n=87). The most commonly detected ARG was OXA-48 (n=93). In addition, we found overnight incubation in non-selective enrichment broth (BPW) to promote the growth of bacteria harbouring extended spectrum beta lactamase (ESBL) genes and reduction in the detection of carbapenemase genes.ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding DNA extraction method for the detection of ARGs isolated from river water samples.

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AN IMPROVED EXTRACTION METHOD TO INCREASE DNA YIELD FROM MOLTED FEATHERS

The Condor ◽

10.1525/cond.2008.8586 ◽

2008 ◽

Vol 110 (4) ◽

pp. 762-766 ◽

Cited By ~ 24

Author(s):

SHELLEY BAYARD DE VOLO ◽

RICHARD T. REYNOLDS ◽

MARLIS R. DOUGLAS ◽

MICHAEL F. ANTOLIN

Keyword(s):

Extraction Method ◽

Dna Yield

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Choice of DNA extraction method affects DNA metabarcoding of unsorted invertebrate bulk samples

Metabarcoding and Metagenomics ◽

10.3897/mbmg.2.26664 ◽

2018 ◽

Vol 2 ◽

Cited By ~ 14

Author(s):

Markus Majaneva ◽

Ola H. Diserud ◽

Shannon H.C. Eagle ◽

Mehrdad Hajibabaei ◽

Torbjørn Ekrem

Keyword(s):

Dna Extraction ◽

Extraction Method ◽

Target Material ◽

Cost Effective ◽

Community Analysis ◽

Extraction Methods ◽

Dna Recovery ◽

Dna Yield ◽

Dna Extraction Method ◽

Dna Metabarcoding

Characterisation of freshwater benthic biodiversity using DNA metabarcoding may allow more cost-effective environmental assessments than the current morphological-based assessment methods. DNA metabarcoding methods where sorting or pre-sorting of samples are avoided altogether are especially interesting, since the time between sampling and taxonomic identification is reduced. Due to the presence of non-target material like plants and sediments in crude samples, DNA extraction protocols become important for maximising DNA recovery and sample replicability. We sampled freshwater invertebrates from six river and lake sites and extracted DNA from homogenised bulk samples in quadruplicate subsamples, using a published method and two commercially available kits: HotSHOT approach, Qiagen DNeasy Blood & Tissue Kit and Qiagen DNeasy PowerPlant Pro Kit. The performance of the selected extraction methods was evaluated by measuring DNA yield and applying DNA metabarcoding to see if the choice of DNA extraction method affects DNA yield and metazoan diversity results. The PowerPlant Kit extractions resulted in the highest DNA yield and a strong significant correlation between sample weight and DNA yield, while the DNA yields of the Blood & Tissue Kit and HotSHOT method did not correlate with the sample weights. Metazoan diversity measures were more repeatable in samples extracted with the PowerPlant Kit compared to those extracted with the HotSHOT method or the Blood & Tissue Kit. Subsampling using Blood & Tissue Kit and HotSHOT extraction failed to describe the same community in the lake samples. Our study exemplifies that the choice of DNA extraction protocol influences the DNA yield as well as the subsequent community analysis. Based on our results, low specimen abundance samples will likely provide more stable results if specimens are sorted prior to DNA extraction and DNA metabarcoding, but the repeatability of the DNA extraction and DNA metabarcoding results was close to ideal in high specimen abundance samples.

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Effects of Plant Tissue and DNA Purification Method on Randomly Amplified Polymorphic DNA-based Genetic Fingerprinting Analysis in Carrot

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.124.1.32 ◽

1999 ◽

Vol 124 (1) ◽

pp. 32-38 ◽

Cited By ~ 66

Author(s):

L.S. Boiteux ◽

M.E.N. Fonseca ◽

P.W. Simon

Keyword(s):

Dna Extraction ◽

Dna Cleavage ◽

Extraction Method ◽

Extraction Methods ◽

The Other ◽

Dna Purification ◽

Genetic Fingerprinting ◽

Purification Method ◽

Fingerprinting Analysis ◽

Dna Yield

Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting analysis using six tissues obtained from inbred carrot (Daucus carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DNA cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and purification methods for the total amount of DNA. An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when compared with the other six nonorganic extraction methods. Of the tissues examined, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fresh tissue); followed by seeds (54 ng·mg-1), fresh leaves (48 ng·mg-1), lyophilized leaves (40 ng·mg-1), calli (22 ng·mg-1), and tap roots (4 ng·mg-1). For most of the preparations, the DNA showed no traces of degradation. However, DNA preparations were not consistently accessible to HindIII cleavage in all tissue-extraction method combinations. Uncut DNA was observed chiefly in extractions from flowers and fresh leaves suggesting a tissue-specific adverse effect on restriction endonuclease activity. Differences in RAPD band (amplicon) intensity and number were observed across tissues and DNA extraction methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD-based fingerprinting yielding a consistently higher number of more intense amplicons when compared to the other tissues. In flowers and seeds, only DNA obtained with the CTAB extraction method could be amplified. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. Judicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for reliable RAPD-based DNA fingerprinting analysis in carrot. In addition, our studies allowed the identification of a better combination of procedures for use in routine manipulations of carrot DNA such as RFLP-RAPD-based cultivar fingerprinting, molecular mapping, screening of transgenic plants, construction of genomic libraries, and gene cloning.

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ANALYSIS OF FOUR DIFFERENT METHODS OF DNA EXTRACTION FROM THE TRUE WEEVIL (COLEOPTERA: CURCULIONIDAE)

FLORA AND FAUNA ◽

10.33451/florafauna.v24i1pp135-138 ◽

2018 ◽

Vol 24 (1) ◽

Author(s):

DIVYA SHARMA ◽

DALIP KUMAR ◽

RHITOBAN RAY CHOUDHURY

Keyword(s):

Dna Extraction ◽

Genomic Dna ◽

Extraction Method ◽

Sitophilus Oryzae ◽

Potassium Acetate ◽

Single Specimen ◽

High Quality ◽

Dna Yield ◽

Yield And Quality ◽

Dna Extraction Method

PCR-based markers have been widely used for the analysis of genetic diversity and to avoid ambiguity, molecular characterization is very effective tool for accurate discrimination and identification of a species in insects. Because these studies require analysis of large number of samples, a DNA extraction method that is fast, inexpensive and yields high quality DNA from the preserved samples, needs to be evaluated. A comparative analysis of four methods for DNA extraction from a single specimen of rice weevil, Sitophilus oryzae preserved in 90% alcohol has been communicated. Significantly higher DNA yields were obtained by using SDS-Potassium acetate method followed by CTAB, DNA XPress and Bioline Isolate II genomic DNA kit. Maximum purity (A260/A280- 1.8) was obtained with Bioline Isolate II genomic DNA kit method. The Absorbance ratio was appreciably low with DNA Xpress kit showing the presence of proteins. Bioline Isolate II genomic DNA kit was time efficient and yielded good quality DNA but at a high cost. Based on DNA yield and quality, these evaluations provide a guide for choosing Bioline Isolate II genomic DNA kit method of DNA extraction for rice weevils and optimizing the extraction conditions for rice weevils.

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DNA Extraction from Bronchial Aspirates for Molecular Cytology: Which Method to Take?

Analytical Cellular Pathology ◽

10.1155/2003/354796 ◽

2003 ◽

Vol 25 (2) ◽

pp. 83-88 ◽

Cited By ~ 7

Author(s):

Hans Jürgen Grote ◽

Viola Schmiemann ◽

Mario Sarbia ◽

Alfred Böcking

Keyword(s):

Success Rate ◽

Dna Extraction ◽

Extraction Method ◽

Globin Gene ◽

Extraction Methods ◽

High Quality ◽

High Molecular Weight Dna ◽

Dna Yield ◽

Extraction Protocol ◽

Dna Extraction Method

Objective: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods.Methods: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated.Results: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of β‐globin gene fragments (268, 536, and 989 bp) was 100%. Conclusions: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR‐based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.

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