Comparative Analysis of the Genomic DNA Isolation Methods on &lt;i&gt;Inula&lt;/i&gt; sp. (Asteraceae)

Emre Sevi̇ndi̇k,Zehra Tuğba Murathan,Fatih Coşkun,Veysel Uzun,Mehmet Yavuz Paksoy

doi:10.15835/nsb849881

Abstract

Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma) DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280) that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R) and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF) did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method.

Highlights

Polymerase chain reactions have brought about radical changes in molecular biology and genetic studies for plants, animal and many microorganisms
When the gel images obtained from DNA isolations were examined, it was seen that the DNA bands obtained through phenol chloroform isoamyl alcohol method were brighter compared to the ones, which were obtained through commercial kit (SIGMA) method
In this study, two different genomic DNA isolation methods were applied on 12 taxa of Inula genus that is distributed throughout Turkey, and Polymerase Chain Reaction (PCR) amplification was performed using primers specific to ITS, trnL-F and ndhF regions

Summary

Introduction

Polymerase chain reactions have brought about radical changes in molecular biology and genetic studies for plants, animal and many microorganisms. It was revealed that these substances bind to nucleic acids firmly during DNA isolation and inhibit Taq- polymerase enzyme and block PCR amplification (Fang et al, 1992; Pirttila et al., 2001; Kermekchıev et al, 2009). For this reason, it is essential to obtain DNA material to be used in the studies within a short time and in a pure form. Various chemicals must be used in different amounts in order to apply these methods (Bailes et al, 2007)

Objectives

Methods

Results

Conclusion