Plant DNA Extraction and Purification Using Nanocomposites

Tetrahymena pyriformis (protozoa) is intensely investigated as a model organism, offering numerous advantages in comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is a vital step of any molecular experiment, here a new mixed surfactant (Sodium dodecyl sulfate (SDS) 20%/Triton X-100) was adopted for effective DNA extraction from Tetrahymena pyriformis under an easy, fast protocol. The efficiency of this technique was then compared with three widely-used alternative techniques, namely the Chelex 100 matrix, Ammonium pyrrolidine dithiocarbamate (APD) complex and SDS–chloroform methods. DNA extraction was analyzed by pulsed-field gel electrophoresis, spectral measurement, fluorometry (Qubit), restriction enzyme digestion, and polymerase chain reaction. Data analysis revealed that the quantity and quality of the recovered DNA varied depending on the applied DNA extraction method. The new method (SDS 20%/Triton X-100) was the most efficient for extracting DNA from Tetrahymena pyriformis with high integrity and purity, affordable cost, less time, and suitability for molecular applications.

Download Full-text

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

BioTechniques ◽

10.2144/btn-2020-0065 ◽

2020 ◽

Vol 69 (4) ◽

pp. 270-280 ◽

Cited By ~ 1

Author(s):

Mustafa Ahmad Munawar ◽

Frank Martin ◽

Anna Toljamo ◽

Harri Kokko ◽

Elina Oksanen

Keyword(s):

Reaction Kinetics ◽

Dna Extraction ◽

Pcr Primers ◽

Sybr Green ◽

Recombinase Polymerase Amplification ◽

Pcr Inhibitors ◽

Plant Dna ◽

Pcr Assays ◽

Flanking Regions

DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid ‘RPA-PCR couple’ concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry Phytophthora pathogens and the Phytophthora identification marker atp9-nad9. We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples.

Download Full-text

Effects of DNA Extraction and Purification Methods on Real-Time Quantitative PCR Analysis of Roundup Ready Soybean

Journal of AOAC International ◽

10.1093/jaoac/92.4.1136 ◽

2009 ◽

Vol 92 (4) ◽

pp. 1136-1144 ◽

Cited By ~ 18

Author(s):

Tigst Demeke ◽

Indira Ratnayaka ◽

Anh Phan

Keyword(s):

Real Time ◽

Dna Extraction ◽

Quantitative Pcr ◽

Real Time Pcr ◽

Roundup Ready ◽

Dna Recovery ◽

Real Time Quantitative Pcr ◽

Extraction And Purification ◽

Purification Methods ◽

Accuracy And Repeatability

Abstract The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

Download Full-text

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays

Journal of Microbiological Methods ◽

10.1016/j.mimet.2005.02.021 ◽

2005 ◽

Vol 63 (2) ◽

pp. 115-126 ◽

Cited By ~ 57

Author(s):

K. Lemarchand ◽

F. Berthiaume ◽

C. Maynard ◽

J. Harel ◽

P. Payment ◽

...

Keyword(s):

Dna Extraction ◽

Pathogen Detection ◽

Extraction And Purification ◽

Wastewater Samples ◽

Microbial Dna ◽

Raw Wastewater

Download Full-text

Rapid and Simple Detection of Trichosporon asahii by Optimized Colony PCR

BioMed Research International ◽

10.1155/2019/1803278 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9

Author(s):

Dequan Zhang ◽

Xuelian Lu ◽

Yong Liao ◽

Zhikuan Xia ◽

Zhuoying Peng ◽

...

Keyword(s):

Dna Extraction ◽

Intergenic Spacer ◽

Glass Beads ◽

Clinical Samples ◽

Trichosporon Asahii ◽

Colony Pcr ◽

Extraction And Purification ◽

Simple Detection ◽

Mock Infection ◽

Species Specific

Trichosporon asahii is the major pathogen causing invasive trichosporonosis. Conventional methods of its detection are time-consuming or costly and often require complex DNA extraction and purification steps, which hinders rapid clinical diagnosis. In this study, we evaluated colony PCR, which directly uses colonies or trace clinical samples as the template for amplification, for rapid detection of T. asahii infection. Four methods, namely, direct colony, freeze-thaw, glass beads, and enzymolysis, were compared to select the best DNA extraction strategy. We subsequently designed and screened species-specific primers targeting the intergenic spacer 1 (IGS1) of the ribosomal DNA of T. asahii and used them to detect mock infection clinical samples. The species-specific colony PCR based on glass beads proved advantageous, with short procedure time (154.8 ± 0.6 min), good sensitivity (detection limit, 102 CFU/mL), and specificity for T. asahii, indicating that this method can be used for the rapid and simple identification of clinical samples of T. asahii infection.

Download Full-text