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

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%.

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.

A modified direct PCR amplification method using the GlobalFiler™ PCR Amplification Kit on bloodstains collected using microFLOQ™ direct swabs

2019 ◽  
Vol 7 (1) ◽  
pp. 30-32
Author(s):  
Yongxun Wong ◽  
Boon Kiat Ng ◽  
Kevin Wai Yin Chong ◽  
Wei Siong Holden Lim ◽  
Afiqah Razanah Rosli ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Direct Pcr ◽  
Amplification Method

A universal DNA extraction and PCR amplification method for fungal rDNA sequence-based identification

Mycoses ◽  
2014 ◽  
Vol 57 (10) ◽  
pp. 612-622 ◽  
Author(s):  
A. M. Romanelli ◽  
J. Fu ◽  
M. L. Herrera ◽  
B. L. Wickes
Keyword(s):  
Dna Extraction ◽  
Pcr Amplification ◽  
Rdna Sequence ◽  
Amplification Method

Simple ‘universal’ DNA extraction procedure compatible with direct PCR amplification

1996 ◽  
Vol 52 (4) ◽  
pp. 295-295 ◽  
Author(s):  
D. Goldenberger ◽  
I. Perschil ◽  
M. Ritzler ◽  
M. Altwegg
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Pcr Amplification ◽  
Direct Pcr

scholarly journals ALKALINE LYSIS AS AN EFFICIENT AND ECONOMICAL ALTERNATIVE FOR DNA EXTRACTION FROM HORSEHAIR HAIR SAMPLES: COMPARISON BETWEEN DIFFERENT METHODS

2021 ◽  
Vol 9 (02) ◽  
pp. 836-841
Author(s):  
Myriam Janeth Ortega Torres ◽  
◽  
Jessica Almeida Braga ◽  
Camilo Torres ◽  
Ahmed Sami Shaker ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Forensic Population ◽  
Alkaline Lysis ◽  
Hair Samples ◽  
Genomic Studies ◽  
Dna Extraction Methods ◽  
Short Time

Studies related to DNA extraction are becoming more ambitious in the sense that large studies are intended to be carried out with minimum DNA sources. The DNA extracted must be of quality for genetic, forensic, population and genomic studies, these samples must be easy to obtain and product of efficient manipulation.Samples obtained from horsehair are an important technical challenge since they constitute the preferred non-invasive sample for genetic studies in horses, which has been shown to obtain reliable results in a short time. In this sense, working into effective techniques to optimizeDNA extraction of scarse samples is a pertinent task. In this study, different DNA extraction methods were evaluated from mane samples obtained from a population of wild horses from the Region of Arauca in eastern Colombia.Three DNA extraction methods were evaluated (phenol chloroform, alkaline lysis and twocommercial DNA extraction kit), DNA concentration, purity and qualityweredeterminate and PCR amplification product were obtain using primers for a hypervariable region of DNA mitochondrial. DNA preparation from hair roots using alkaline lysis was the most economical and efficient method with which it was possible to obtain high quality and quantity DNA.

scholarly journals Can non-destructive DNA extraction of bulk invertebrate samples be used for metabarcoding?

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4980 ◽  
Author(s):  
Melissa E. Carew ◽  
Rhys A. Coleman ◽  
Ary A. Hoffmann
Keyword(s):  
Dna Extraction ◽  
Dna Barcode ◽  
Extraction Methods ◽  
Biological Communities ◽  
Freshwater Invertebrate ◽  
High Throughput Dna Sequencing ◽  
Dna Extraction Methods ◽  
Taxonomic Groups ◽  
Taxonomic Studies ◽  
Non Destructive

Background High throughput DNA sequencing of bulk invertebrate samples or metabarcoding is becoming increasingly used to provide profiles of biological communities for environmental monitoring. As metabarcoding becomes more widely applied, new reference DNA barcodes linked to individual specimens identified by taxonomists are needed. This can be achieved through using DNA extraction methods that are not only suitable for metabarcoding but also for building reference DNA barcode libraries. Methods In this study, we test the suitability of a rapid non-destructive DNA extraction method for metabarcoding of freshwater invertebrate samples. Results This method resulted in detection of taxa from many taxonomic groups, comparable to results obtained with two other tissue-based extraction methods. Most taxa could also be successfully used for subsequent individual-based DNA barcoding and taxonomic identification. The method was successfully applied to field-collected invertebrate samples stored for taxonomic studies in 70% ethanol at room temperature, a commonly used storage method for freshwater samples. Discussion With further refinement and testing, non-destructive extraction has the potential to rapidly characterise species biodiversity in invertebrate samples, while preserving specimens for taxonomic investigation.

Comparison of DNA extraction methods from muscle of canned tuna for species identification

Food Control ◽  
2007 ◽  
Vol 18 (10) ◽  
pp. 1211-1215 ◽  
Author(s):  
María José Chapela ◽  
Carmen G. Sotelo ◽  
Ricardo I. Pérez-Martín ◽  
Miguel Ángel Pardo ◽  
Begoña Pérez-Villareal ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Species Identification ◽  
Extraction Methods ◽  
Canned Tuna ◽  
Dna Extraction Methods

scholarly journals DNA Barcoding Analysis and Phylogenetic Relation of Mangroves in Guangdong Province, China

Forests ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 56 ◽  
Author(s):  
Feng Wu ◽  
Mei Li ◽  
Baowen Liao ◽  
Xin Shi ◽  
Yong Xu
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Dna Barcode ◽  
Phylogenetic Reconstruction ◽  
Pcr Amplification ◽  
Guangdong Province ◽  
Nuclear Ribosomal Dna ◽  
Success Rates ◽  
Mangrove Species ◽  
Identification Rate

Mangroves are distributed in the transition zone between sea and land, mostly in tropical and subtropical areas. They provide important ecosystem services and are therefore economically valuable. DNA barcoding is a useful tool for species identification and phylogenetic reconstruction. To evaluate the effectiveness of DNA barcoding in identifying mangrove species, we sampled 135 individuals representing 23 species, 22 genera, and 17 families from Zhanjiang, Shenzhen, Huizhou, and Shantou in the Guangdong province, China. We tested the universality of four DNA barcodes, namely rbcL, matK, trnH-psbA, and the internal transcribed spacer of nuclear ribosomal DNA (ITS), and examined their efficacy for species identification and the phylogenetic reconstruction of mangroves. The success rates for PCR amplification of rbcL, matK, trnH-psbA, and ITS were 100%, 80.29% ± 8.48%, 99.38% ± 1.25%, and 97.18% ± 3.25%, respectively, and the rates of DNA sequencing were 100%, 75.04% ± 6.26%, 94.57% ± 5.06%, and 83.35% ± 4.05%, respectively. These results suggest that both rbcL and trnH–psbA are universal in mangrove species from the Guangdong province. The highest success rate for species identification was 84.48% ± 12.09% with trnH-psbA, followed by rbcL (82.16% ± 9.68%), ITS (66.48% ± 5.97%), and matK (65.09% ± 6.00%), which increased to 91.25% ± 9.78% with the addition of rbcL. Additionally, the identification rate of mangroves was not significantly different between rbcL + trnH-psbA and other random fragment combinations. In conclusion, rbcL and trnH-psbA were the most suitable DNA barcode fragments for species identification in mangrove plants. When the phylogenetic relationships were constructed with random fragment combinations, the optimal evolutionary tree with high supporting values (86.33% ± 4.16%) was established using the combination of matK + rbcL + trnH-psbA + ITS in mangroves. In total, the 476 newly acquired sequences in this study lay the foundation for a DNA barcode database of mangroves.

Extraction and amplification of DNA from aged and archaeological Populus euphratica wood for species identification

Holzforschung ◽  
2015 ◽  
Vol 69 (8) ◽  
pp. 925-931 ◽  
Author(s):  
Lichao Jiao ◽  
Xiaoli Liu ◽  
Xiaomei Jiang ◽  
Yafang Yin
Keyword(s):  
Species Identification ◽  
Arid Regions ◽  
Wood Specimen ◽  
Dna Barcode ◽  
Populus Euphratica ◽  
Pcr Amplification ◽  
The Other ◽  
Western China ◽  
Archaeological Wood ◽  
Plant Remains

Abstract The wood samples of Populus euphratica Oliv. (Salicaceae) are common archaeological plant remains in the hot and arid regions of western China. However, it is difficult to identify P. euphratica wood based on traditional wood anatomical methods alone. DNA barcoding might provide a higher security for species identification. In this study, aged wood specimens stored for approximately 30, 60, and 80 years and archaeological wood up to 3600 years old were in focus to explore the potential of DNA extraction and PCR amplification for different-sized fragments, ranging between 100 and 800 bp, taken from wood stored for different periods. The results indicated that DNA fragments of more than 100 bp could be successfully retrieved from a wood specimen stored for about 80 years based on a modified Qiagen kit protocol. However, it was impossible to obtain DNA segments from the 3600-year-old wood according to the current extraction protocol. Moreover, it was deduced that two-stage PCR amplification could play a significant role in the analysis of DNA retrieved from aged wood materials. With the aid of phylogenetic analysis, based on the short DNA barcode rbcL-2 of 202 bp in length, it was possible to differentiate P. euphratica from the other species of the Populus genus.

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