A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification.

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser®. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.Key words: conifers, wood, polymerase chain reaction, sequencing.

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Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains

Stamford Journal of Pharmaceutical Sciences ◽

10.3329/sjps.v4i1.8867 ◽

1970 ◽

Vol 4 (1) ◽

pp. 53-57 ◽

Cited By ~ 12

Author(s):

Mohammad Shahriar ◽

Md Rashidul Haque ◽

Shaila Kabir ◽

Irin Dewan ◽

Mohiuddin Ahmed Bhuyian

Keyword(s):

Bacillus Subtilis ◽

Dna Extraction ◽

Genomic Dna ◽

Extraction Procedure ◽

Proteinase K ◽

Clinical Samples ◽

Gram Positive ◽

Genomic Dna Extraction ◽

Dna Concentration ◽

The Mean

Direct extraction of DNA from natural environment and clinical samples has become a useful alternative for the phylogenetic identification and in situ detection of individual microbial cells without cultivation. In this study, three different Gram positive microorganisms (B. cereus, B. subtilis, and S. aureus) were chosen for genomic DNA extraction. High salt SDS (Sodium Dodesyl Sulfate) based extraction method was followed to extract genomic DNA with addition of three different lysis protocols to observe the effect of proteinase-K on total genomic DNA yield, lysis steps were carried with SDS, SDS with 3 μl proteinase-K and SDS with 6μl proteinase-K. High molecular weight intact DNA bands were observed only for Bacillus subtilis when the extraction procedure was carried out in presence of SDS, SDS with proteinase-K (3μl) and SDS with increased amount of proteinase-K (6μl). In presence of SDS and increased amount of proteinase-K (6μl) the mean value of DNA concentration for Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus were found to be 1.53±0.15, 1.36±0.10 and 1.65±0.10 μg/μl respectively. However, in absence of proteinase-K, the mean values of DNA concentration were found to be decreased (1.28±0.10, 1.34±0.15, 1.23±0.10 μg/μl for B. cereus, B. subtilis, and S. aureus respectively) for all these stains. Although in case of B. subtilis the overall effect of proteinase-K was not found to be significant in terms of DNA concentration and DNA band intensity, however, for B. cereus, and S. aureus sharp decrease in total extracted DNA concentration was observed suggesting the increased lysis effect of proteinase-K on the thick peptidoglycan layer of Gram-positive cell wall such as B. cereus, and S. aureus. Key words: Extraction; Genomic DNA; Lysis buffer; Gram positive organism. DOI: http://dx.doi.org/10.3329/sjps.v4i1.8867 SJPS 2011; 4(1): 53-57

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Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

Methods and Protocols ◽

10.3390/mps2020036 ◽

2019 ◽

Vol 2 (2) ◽

pp. 36

Author(s):

Lorena G. Caligiuri ◽

Adolfo E. Sandoval ◽

Jose C. Miranda ◽

Felipe A. Pessoa ◽

María S. Santini ◽

...

Keyword(s):

Dna Extraction ◽

Internal Control ◽

Large Scale ◽

Pcr Amplification ◽

Proteinase K ◽

Sand Fly ◽

Regulatory Function ◽

Sand Flies ◽

Lysis Buffer ◽

Commercial Kits

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

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Comparison of direct PCR and PCR amplification after DNA extraction for the detection of viable enterotoxigenic Escherichia coli in laboratory microcosms

Journal of Microbiological Methods ◽

10.1016/0167-7012(96)00817-2 ◽

1996 ◽

Vol 26 (1-2) ◽

pp. 21-26 ◽

Cited By ~ 4

Author(s):

Zohreh Tamanai-Shacoori ◽

Anne Jolivet-Gougeon ◽

Michel Cormier

Keyword(s):

Escherichia Coli ◽

Dna Extraction ◽

Pcr Amplification ◽

Enterotoxigenic Escherichia Coli ◽

Direct Pcr

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Optimisation of DNA Extraction from Individual Sand Flies for PCR Amplification

10.20944/preprints201812.0189.v1 ◽

2018 ◽

Author(s):

Lorena G. Caligiuri ◽

Adolfo E. Sandoval ◽

Jose C. Miranda ◽

Felipe A. Pessoa ◽

María S. Santini ◽

...

Keyword(s):

Dna Extraction ◽

Internal Control ◽

Large Scale ◽

Pcr Amplification ◽

Personal Communication ◽

Proteinase K ◽

Sand Fly ◽

Regulatory Function ◽

Sand Flies ◽

Lysis Buffer

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analysing parasite infection in Lutzomyia spp. by PCR [1] and, for this reason, we evaluated various modifications on a previously published protocol ([2] and Acardi personal communication). The most significant variation was the use of a different lysis buffer [3] to which added Ca2+ (buffer TESCa), because this ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site [4]. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene [5,6]. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

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A Syringe-Based and Centrifugation-Free DNA Extraction Procedure for the Rapid Detection of Bacteria

Chemosensors ◽

10.3390/chemosensors9070167 ◽

2021 ◽

Vol 9 (7) ◽

pp. 167

Author(s):

Taehwi Yoon ◽

Seokjoon Kim ◽

Jung Ho Kim ◽

Ki Soo Park

Keyword(s):

Nucleic Acid ◽

Dna Extraction ◽

Extraction Procedure ◽

Food Poisoning ◽

Pcr Amplification ◽

High Sensitivity ◽

Diagnostic Systems ◽

Extraction Device ◽

One Step ◽

Polymerase Chain

Several bacteria are known to cause food poisoning; therefore, diagnostic systems that detect bacteria are essential. Nucleic acid-based testing methods that involve polymerase chain reaction (PCR) amplification are of great interest due to their high sensitivity and specificity. Herein, we developed a syringe-based one-step DNA extraction device that streamlines the extraction of genomic DNA (gDNA) from bacteria within 2 min, enabling versatile application of nucleic acid-based testing in the field. Notably, the bolt-nut structured case coupled with the syringe enables control of the volume of solution dispensed for enabling DNA extraction without the need for bulky centrifuge equipment. Using the proposed system, the gDNA of a model bacterium, Escherichia coli, was extracted at a good quantity and quality and amplified via PCR. The DNA extracted was comparable to that extracted via a centrifugation-based procedure. In addition, bacteria that were artificially spiked in common samples, including a work cloth, a work bench, and meat, were successfully detected with high accuracy.

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Comparison and Validation of Ichthyoplankton DNA Extraction Methods

Methods and Protocols ◽

10.3390/mps4040087 ◽

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.

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Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/948/1/012013 ◽

2021 ◽

Vol 948 (1) ◽

pp. 012013

Author(s):

F Fitriyah ◽

Y Faramitha ◽

D A Sari ◽

I Kresnawaty ◽

T Panji ◽

...

Keyword(s):

Dna Extraction ◽

Species Identification ◽

Dna Barcode ◽

Pcr Amplification ◽

Extraction Methods ◽

Rbcl Gene ◽

Direct Pcr ◽

Nannochloropsis Gaditana ◽

Amplification Method ◽

Microalgal Culture

Abstract Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

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Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

International Journal of Bioassays ◽

10.21746/ijbio.2017.04.004 ◽

2017 ◽

Vol 6 (04) ◽

pp. 5347 ◽

Cited By ~ 3

Author(s):

Omar B. Ahmed* ◽

Anas S. Dablool

Keyword(s):

Dna Extraction ◽

Control Method ◽

Extraction Procedure ◽

Cost Effective ◽

High Yield ◽

Gram Negative Bacteria ◽

Bacterial Dna ◽

Gram Negative ◽

E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

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