Protocol for DNA extraction from any plant species (alkaline PVPP method)

O estabelecimento de protocolo de extração de DNA de espécies vegetais é uma técnica empregada para a obtenção de um DNA puro e de qualidade. Diante disso, objetivou-se neste estudo padronizar um protocolo para a extração de DNA da espécie Acianthera ciliata, visando posteriores estudos de diversidade genética. Foram testados dois métodos de trituração do tecido foliar, sendo eles: Tampão STE e nitrogênio líquido. Para cada método de trituração foram testadas duas concentrações de β-mercaptoetanol (0% e 2%). Os dois métodos utilizados, foram eficientes na extração do DNA genômico de A. ciliata. As amostras extraídas com 0% de β-mercaptoetanol, para os dois métodos, STE e nitrogênio líquido, apresentaram menor quantidade de DNA quando comparado com as amostras extraídas com 2% de β-mercaptoetanol. Os dois primers testados amplificaram regiões do genoma de A. ciliata. Para a extração de DNA de A. ciliata indica-se a utilização de CTAB 5% no tampão de extração e β-mercaptoetanol a 2%. Os iniciadores ISSR foram eficientes na amplificação e são recomendados para estudos de diversidade genética de A. ciliata.Palavras-chave: diversidade genética; CTAB; marcadores moleculares; orquídeas. EVALUATION OF TWO MACERATION METHODS IN Acianthera ciliata (Orchidaceae) LEAVES FOR DNA EXTRACTION ABSTRACT: The establishment of DNA extraction protocol for plant species is a technique employed to obtain pure and good quality DNA. In this study, we standardized a protocol for the extraction of DNA of the species Acianthera ciliata, aiming studies of genetic diversity subsequently. Two maceration methods for foliar tissue were tested, and they were STE buffer and liquid nitrogen. Two concentrations of β-mercaptoethanol (0% and 2%) were tested for each method. The two methods used were efficient for genomic DNA extraction of A. ciliata. In both methods the samples extracted using 0% of β-mercaptoethanol, they presented lesser amount of DNA than the samples extracted using 2% of β-mercaptoethanol. The two tested primers amplified genomic regions of A. ciliata. For the DNA extraction of A. ciliata, we indicated the use of CTAB 5% in the extraction buffer as well as β-mercaptoethanol to 2%. The ISSR primers were efficient in amplification and thus they are indicated for studies of genetic diversity of A. ciliata.Keywords: genetic diversity; CTAB; molecular markers; orchids.

Download Full-text

A high throughput DNA extraction method from chemotypically heterogeneous plant species

Protocol Exchange ◽

10.1038/protex.2013.018 ◽

2013 ◽

Cited By ~ 4

Author(s):

Parthadeb Ghosh ◽

Sanjib Kumar Chattopadhyay ◽

Sinchan Adhikari ◽

Soumen Saha ◽

...

Keyword(s):

Dna Extraction ◽

Plant Species ◽

High Throughput ◽

Extraction Method ◽

Dna Extraction Method

Download Full-text

Evaluation of Two New Methods for DNA Extraction of “Legal High” Plant Species

Journal of Forensic Sciences ◽

10.1111/1556-4029.14478 ◽

2020 ◽

Vol 65 (5) ◽

pp. 1704-1708

Author(s):

Angelique L. Ryan ◽

Cassandra P. O’Hern ◽

Kelly M. Elkins

Keyword(s):

Dna Extraction ◽

Plant Species ◽

New Methods ◽

Legal High

Download Full-text

Rapid and Efficient DNA Extraction Method from Various Plant Species Using Diatomaceous Earth and a Spin Filter

Breeding Science ◽

10.1270/jsbbs.52.151 ◽

2002 ◽

Vol 52 (2) ◽

pp. 151-155 ◽

Cited By ~ 22

Author(s):

Junichi Tanaka ◽

Shigeru Ikeda

Keyword(s):

Dna Extraction ◽

Plant Species ◽

Extraction Method ◽

Diatomaceous Earth ◽

Spin Filter ◽

Dna Extraction Method

Download Full-text

Plant Genomic DNA Extraction for Selected Herbs and Sequencing their Internal Transcribed Spacer Regions Amplified by Specific Primers

Natural Product Communications ◽

10.1177/1934578x1601101017 ◽

2016 ◽

Vol 11 (10) ◽

pp. 1934578X1601101

Author(s):

Farah Izana Abdullah ◽

Lee Suan Chua ◽

Zaidah Rahmat ◽

Azman Abd Samad ◽

Alina Wagiran

Keyword(s):

Dna Extraction ◽

Plant Species ◽

Dna Sequences ◽

Incubation Time ◽

Internal Transcribed Spacer ◽

Genomic Dna ◽

Molecular Technique ◽

Hexadecyltrimethylammonium Bromide ◽

Genomic Dna Extraction ◽

Dna Concentration

This study was focused on plant genomic DNA extraction and sequencing from five commonly used medicinal herbs, namely Impatiens balsamina, Ficus deltoidea, Centella asiatica, Andrographis paniculata and Orthosiphon aristatus. This molecular technique is another highly reliable alternative for plant species identification besides phytochemical profiling. Three cetyl hexadecyltrimethylammonium bromide (CTAB) based methods with slight modification on incubation time, salt content and other additives were used for DNA extraction. The CTAB method of Doyle and Doyle produced higher DNA concentration from I. balsamina, most probably due to the presence of ammonium acetate in the washing buffer and longer incubation time (2 h). The CTAB based method was suitable for A. paniculata because a high DNA concentration of acceptable quality was obtained for all the modified methods. However, O. aristatus was likely to have a lower DNA concentration (33–87 μg/g) and quality, probably due to the high concentration of phenolic compounds, particularly rosmarinic acid. The extracted genomic DNA was effectively amplified by a polymerase chain reaction using a universal primer of internal transcribed spacer (ITS), particularly AB101 and AB102 at the optimum annealing temperature of 48°C. The DNA sequences were analyzed by phenetic analysis and it was found that they have high similarity with the nucleotide sequences of ITS regions for similar plant species in the GenBank database of the National Center for Biotechnology Information.

Download Full-text