Two methods of DNA extraction and purification in eggplant molecular research were investigated. FTA PlantSaver card and DNAzol were optimized, simplified and presented. The aim was to discard the rigorous protocols and help save energy, resources and time wasted in DNA extraction during molecular research in eggplant. DNA was extracted from fresh leaf of 18-day-old eggplant seedlings. Quality of genomic DNA extracts was verified on 1% agarose gel electrophoresis and viewed on a UV-transilluminator. DNA extracted from nine (9) different accessions was PCR amplified using nine (9) RAPD primers. Results indicated that both methods were effective in extracting and purifying sufficient quantity of DNA under different requirements. DNAzol method required the use of more leaf samples to extract sufficient quantity of DNA which in turn required that more seeds should be sown per accession. However, sufficient quantity of DNA could be extracted from even a single leaf sample using the FTA card method which may be preferred in a situation of low seed viability. Therefore, FTA card method is recommended for timely and quality DNA extractions and amplification using simple protocols outlined in this article.
Research in plant molecular biology starts with nucleic acid isolation and purification followed by amplification [
Research activities were carried out in the Molecular Biology Laboratory of the University of Agriculture, Makurdi Nigeria. Eggplant seeds of nine accessions were planted in small transparent containers labeled D1-D9 respectively. DNA was extracted from fresh and healthy leaf samples (18-day-old) using FTA PlantSaver card and DNAzol methods. Complex protocols were continuously subjected to modification until optimized and simpler procedures were achieved in extracting sufficient quantity of DNA. Quality of genomic DNA extracts was verified on 1% agarose gel electrophoresis. Images were captured on the UV-transilluminator. Other extractions were immediately followed by PCR amplification of nine DNAs labeled D1-D9 using nine (9) RAPD primers: OPP-11, OPV-04, OPQ-07, OPU-19, OPQ-03, V-19, B-18, OPU-13 and OPU-15 respectively. Amplification was achieved on a thermal cycler (Applied Biosystem version) where 1 µl of DNA extracted from DNAzol or 1 purified FTA disc served as the DNA template in the reactions. Amplified products were resolved on 3% agarose gel electrophoresis and viewed on the UV-transilluminator. All results were compared accordingly.
DNA extraction is the first step in the study of plant DNA as it precedes all other molecular activities [
Analysis of the DNAzol method shows that more leaf samples are needed to achieve 1 g pulverized leaf tissue required. This suggests that more viable seeds should be planted (
The use of FTA card method, on the other hand, is recommended when at least one leaf sample is required. It is often employed in a situation of low seed viability. At least, one seedling is sufficient enough for DNA extraction. It is a simpler method than DNAzol but requires repeated DNA washing. It does not consume space as one card can be used for a maximum of eight accessions when the squares are properly demarcated [
DNAzol method | FTA PlantSaver card method |
---|---|
Materials: Fresh and healthy leaf sample (15 - 21 day old); Mortar and pestle; DNAZOL reagent; Absolute ethanol; 70% ethanol; DNAzol-ethanol wash (optional); Chloroform timer; Micropipettes; 1.5 ml microcentrifuge tubes; Vortex machine; Centrifuge machine; Freezer, plastic rack, hand gloves and lab coat. | Materials: Fresh and healthy leaf sample (15 - 21 day old); FTA PlantSaver card; PestleParafilm paper; Harris punch; Harris cutting mat; Cotton wool; Desiccator; Absolute ethanol; FTA purification reagent; 70% ethanol; Timer; Micropipettes; 1.5 ml microcentrifuge tubes; Vortex machine; Plastic rack; Freezer; Hand gloves and lab coat. |
Label your microcentrifuge tube (1.5 ml - 2 ml capacity) with a number representing the accession code. | Place a leaf sample in a labeled square of the FTA card. |
Weigh 1 g of leaf sample and place in a mortar. Add 5 ml of absolute ethanol to submerge the leaf tissue for 30 minutes. Decant excess ethanol. | Overlay the sample with a transparent parafilm. |
Dispense 750 µl DNAzol reagent into the tube | Gently pound the leaf until greenish sap is transferred beneath the paper. |
Pulverize the leaf tissue in the mortar. | Remove the parafilm and air dry the card for 1 hour. |
Transfer the homogenize tissue to the tube containing DNAzol. Allow the mixture stand for 5 minutes. | Cut 2 discs (2 mm diameter) from the sample into a 1.5 ml tube using the Harris punch. |
Add 750 µl chloroform to the mixture above. Allow the mixture stand for 5 minutes. | Add 200 µl of 70% ethanol to the tube and allow soaking for 5 minutes. Vortex for 30 minutes before discarding the liquid, leaving the discs in the tube. |
Centrifuge the tube at 10,000× g for 10 minutes. | Repeat the last step. |
Transfer the supernatant (portion containing the DNA) into a new and labeled tube. | Dispense 200 µl of FTA purification reagent to the tube; allow to soak for 5 minutes. Vortex for 30 minutes before discarding the liquid gently, leaving the discs in the tube. |
Add 750 µl absolute ethanol to the transferred supernatant to precipitate the DNA for 5 minutes. | Repeat the last step. |
Centrifuge at 5000× g for 5 minutes to produce pelletized DNA. | Transfer the two discs containing purified DNA into a fresh tube using micropipette tips. |
Add 750 µl of 70% ethanol to resuspend the pelletized DNA. Allow the mixture to stand for 5 minutes. | Air the discs in the tube for 1 hour. |
Further centrifuge at 5000× g for 5 minutes. | Store in a freezer at −20˚C for further use. |
Gently decant the liquid portion leaving the pelletized and pure DNA extracted. | Each disc can serve as a DNA template for PCR. |
Air dry the tube for 1 hour. Reconstitute the DNA in 100 µl 1× TE for further use. | Comment: FTA method is recommended for timely and quality DNA extraction and amplification from large number of samples. |
strongly recommended for timely and quality DNA extraction and amplification from a large number of samples. Generally, the two optimized protocols are efficient in DNA extraction which may replace the use of complicated protocols. This would help researchers in achieving quality DNA extraction and also help save resources, energy and time. The protocols presented in this report may therefore be used in extracting DNA from other crops.
Celestine Uzoma Aguoru,Lucky O. Omoigui,Joseph Olalekan Olasan, (2015) Comparative Optimized Protocols of DNA Extraction and Purification Using FTA PlantSaver Card and DNAzol Methods for Eggplant (Solanum Species) Studies in North Central Nigeria. Open Access Library Journal,02,1-5. doi: 10.4236/oalib.1101406