DNA isolation of clove and lemongrass using modification of the Doyle and Doyle methods and their relation with antioxidant activity

Abstract

Clove (Syzigium aromaticum) and lemongrass (Cymbopogon winterianus) are essential oil-producing plants that are widely available in Indonesia and have high economic value for medicinal and industrial needs because of their antioxidant activity. However, the information about the relationship between antioxidant activity and the DNA content in these plants was very limited. Especially because DNA isolation of aromatic plants is also not easy to do with methods that are widely used. Therefore, this study aims to isolate DNA from clove and lemongrass using a modified Doyle and Doyle method and to find the relationship between the quality and quantity of DNA with their antioxidant activity. The method used was the use of PVP, isopropanol and sodium acetate for the modification of the Doyle and Doyle method in DNA isolation followed by antioxidant analysis using DPPH. The results showed that the modification to the Doyle and Doyle method had obtained high concentrations of DNA. Besides that, the clove DNA content is higher than lemongrass which is supported by the antioxidant activity of both plants.

What is the “modified” CTAB protocol? Characterizing modifications to the CTAB DNA extraction protocol

“…β‐mercaptoethanol is most commonly added to the CTAB lysis buffer. The concentrations of β‐mercaptoethanol ranged from 0% (Listyanto et al, 2021 ) to 5% (Arruda et al, 2017 ), but most protocols use 0.2–0.5%, which is similar to Doyle and Doyle's ( 1987 ) usage of 0.2%. The Fairlie and Pokorny protocol (cited in Larridon et al, 2020 ) recommended an increase of β‐mercaptoethanol from 0.2% to 0.4% when extracting DNA from herbarium specimens.…”

“…The amount of starting tissue used for DNA extractions varied substantially across protocols. For dried tissue, protocols used anywhere between 0.4–6000 mg (Syamkumar et al, 2003 ; Listyanto et al, 2021 ). Most protocols added 1000 mg of leaf tissue, which is similar to Doyle and Doyle ( 1987 ), who used 500–1500 mg.…”

“…Silva ( 2010 ) experimentally determined that higher concentrations of β‐mercaptoethanol, in their case 1–5%, resulted in cleaner and higher yields of DNA; however, any adverse effects of using high concentrations of β‐mercaptoethanol on downstream reactions are unknown, and further study is needed. β‐mercaptoethanol's toxicity and off‐putting smell has led many laboratories to omit its use in extractions (Listyanto et al, 2021 ); nevertheless, it does serve an important function in plant DNA extractions by degrading and removing proteins and preventing oxidation (Abubakar et al, 2018 ).…”

“…RNase breaks down RNA into ribonucleosides, inhibiting the coprecipitation of RNA with DNA during CTAB extractions (Zamboni et al, 2008 ). Some protocols do not use RNase A (Listyanto et al, 2021 ), but the presence of RNA could interfere with primer sites during PCR amplification (Jobes et al, 1995 ; Porebski et al, 1997 ). The application of RNase A occurred at different steps in the extraction protocol: before the EtOH wash step (Demeke et al, 2009 ) or after the elution stage (Abubakar et al, 2018 ).…”