Optimized CTAB-Based DNA Extraction from Cichorium intybus L.: A Reliable Approach for PCR Applications

Background: Cichorium intybus L. (chicory), a perennial herb of the Asteraceae family, is widely recognized for its medicinal value. However, isolating high-quality DNA from chicory remains challenging due to its rigid cell walls and the abundance of polyphenols and polysaccharides, which interfere with conventional extraction methods, often resulting in low yields and degraded DNA.

Objective: Our study aimed to optimize the cetyltrimethylammonium bromide (CTAB) method for efficient DNA extraction from chicory leaves, followed by validation using polymerase chain reaction (PCR).

Methods: Fresh, etiolated young chicory leaves were collected from four regions of Uttarakhand (India) and stored at -80°C before processing. Cell lysis was performed using pre-warmed 1x CTAB extraction buffer, omitting liquid nitrogen. RNase A treatment at 37°C removed RNA, followed by phase separation with a chloroform/isoamyl alcohol (24:1 v/v) mixture and DNA precipitation using isopropanol and 7.5M ammonium acetate. The extracted DNA was dissolved in 1x TE buffer and stored at -20°C. Yield and purity were assessed using a NanoDrop UV–Vis spectrophotometer, and integrity was confirmed via agarose gel electrophoresis. Primers for the 1-SST gene of chicory were designed using Primer3 and amplified by PCR.

Results: Method efficiency was improved by increasing the concentration of CTAB (2%) and polyvinylpyrrolidone (2%), along with the inclusion of 7.5 M ammonium acetate. The pre-warmed CTAB buffer eliminated the need for liquid nitrogen, simplifying the protocol. DNA yield averaged 64.56 ± 30.51 ng/μl, with an OD_{260/280 nm} of 1.75 ± 0.12. PCR successfully amplified a 2.01 kb fragment of the 1-SST gene in all samples, confirming the protocol’s reliability.

Conclusion: To the best of our knowledge, this is the first tailored protocol for DNA extraction from chicory. Compared to costly commercial kits, the optimized CTAB method is cost-effective, reproducible, and suitable for downstream molecular applications, thus advancing genomic research.