Jul 01, 2025
Isolation of Circular Single-Stranded DNA from Total DNA in Grapevine Samples
- Vahid Jalali Javaran1
- 1Université de Sherbrooke
- Nanovirseq
Protocol Citation: Vahid Jalali Javaran 2025. Isolation of Circular Single-Stranded DNA from Total DNA in Grapevine Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82o38l2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 16, 2024
Last Modified: July 01, 2025
Protocol Integer ID: 107704
Keywords: epigenetic modifications of circular ssdna virus, length sequencing of the circular ssdna virus, native form of circular ssdna virus, circular ssdna virus, viruses from total dna extraction, purified viral dna, grapevine samples this protocol, viral dna, grapevine sample, total dna extraction, stranded dna, dna from total dna, dna, total dna, ssdna, detection of methylation pattern, genome structure, genome, epigenetic modification, methylation pattern
Abstract
This protocol details a method for the purification and enrichment of circular single-stranded DNA (ssDNA) viruses from total DNA extractions. Through a series of enzymatic reactions, the protocol is optimized to selectively enrich the native form of circular ssDNA viruses, preserving chemical modifications such as methylation on their genomes. The expected results include highly purified viral DNA, enabling full-length sequencing of the circular ssDNA virus and the detection of methylation patterns. This makes the protocol ideal for studying both the genome structure and epigenetic modifications of circular ssDNA viruses.
Guidelines
- Prepare all buffers and enzyme mixes fresh before use to ensure optimal enzyme activity.
- Use sterile, DNase-free water and tubes to avoid contamination of the DNA sample.
- Ensure accurate pipetting of enzymes and reaction components, as enzyme activity depends on precise reaction conditions.
- Incubation times and temperatures must be strictly followed for efficient digestion and processing of the DNA.
- Bead washing and elution steps should be performed gently to prevent DNA loss or damage.
Materials
Buffers and Solutions:
rCutSmart‱ Buffer (10X) (Modified as NEBuffer‱ 4 by adding DTT):
- 50 mM Potassium Acetate
- 20 mM Tris-acetate
- 10 mM Magnesium Acetate
- 100 µg/ml Recombinant Albumin
- 1 mM DTT (added to create NEBuffer‱ 4)
- pH 7.9 at 25°C
- E. coli DNA Ligase I Buffer (10X): Used for ligation reactions with DNA ligase and ploymerase activity with Klenow.
- Sodium Acetate (3 M, pH 5.2)
- Cold 75% Ethanol (pre-chilled at -20°C)
- 100% Ethanol
- DNase-free Water
Enzymes:Restriction Enzymes:
- SrfI (20,000 units/ml)
- SpeI-HF (20,000 units/ml)
- Nicking Endonuclease:Nt.AlwI (10000 units/ml)
- Exonucleases:T7 Exonuclease (Double-stranded DNA specific, 10,000 units/ml)
- Exonuclease T (Single-stranded DNA specific, 5,000 units/ml)
- E. coli DNA Ligase (10,000 units/ml)
- DNA Polymerase I, Large (Klenow) Fragment (5,000 units/ml)
Consumables:
- Microcentrifuge tubes (1.5 mL and 2 mL)
- Pipette tips (sterile, DNase-free)
- Magnetic beads for bead washing and DNA elution (AMPure XP Bead-Based Reagent)
Equipment:
- Centrifuge capable of speeds up to 15,700 g
- Magnetic stand for bead washing
- Qubit fluorometer kit for DNA quantification
- PCR machine or thermal cycler
- Heating block or water bath (set to 37°C and 25°C as needed)
Troubleshooting
Safety warnings
- Always wear gloves and use a fume hood when handling enzymes, DTT, and other reagents to avoid contamination and exposure.
- Restriction enzymes and exonucleases are temperature-sensitive; ensure proper storage and handling to maintain activity.
- Dispose of all biohazardous waste, such as used tips and tubes, according to your institution's biosafety protocols.
- Bead washing steps must be performed carefully to prevent DNA loss; do not overdry or overwash the beads.
- Validate digestion efficiency (e.g., Qubit) before proceeding to ensure the protocol's success.
Before start
- Confirm the total DNA concentration is sufficient for the reaction (we used ~22 µg for initial digestion).
- Prepare the following buffers and enzyme mixes:
- rCutSmart‱ Buffer for restriction enzyme digestion.
- E. coli DNA Ligase Reaction Buffer for polymerase activity.
- Preheat the incubators or water baths to 37°C for restriction digestion and 25°C for exonuclease reactions.
- Have bead washing and elution materials ready, ensuring the beads are appropriately equilibrated for efficient DNA recovery.
Double-Stranded Circular DNA Digestion
Prepare the Reaction Mixture:
- Combine the following in a microcentrifuge tube:
| Component | Volume | Details | |
| Total DNA | ~ | ~22 µg total DNA extracted from grapevine samples | |
| rCutSmart Buffer (10X) | 18 µL | Provides optimal conditions for restriction enzyme activity | |
| SrfI | 3 µL | 60 units, cuts specific dsDNA sites | |
| SpeI-HF | 3 µL | 60 units, cuts specific dsDNA sites | |
| Nt.AlwI | 3 µL | 30 units, introduces single-strand breaks into circular dsDNAs | |
| DNase-free water | Adjust to 180 µL | Final reaction volume |
Explanation: This step digests double-stranded circular genomes and plasmids to linearize them, ensuring they can be degraded in the subsequent steps.
Point: The selection of restriction enzymes like SrfI and SpeI-HF in the protocol depends on the specific recognition sites present in the chloroplast and mitochondrial genomes of the plant species being studied.
Incubation: Incubate at 37°C for 2 hours to ensure complete digestion.
Digestion of Linear Double- and Single-Stranded DNAs
This step removes all linear DNA fragments (both double-stranded and single-stranded) that were generated during the restriction digestion step. The remaining DNA will predominantly consist of circular single-stranded DNA (ssDNA).
| Component | Volume | Details | |
| T7 Exonuclease | 3 µL | 30 units; degrades linear double-stranded DNA efficiently | |
| Exonuclease T | 5.1 µL | 25 units; degrades linear single-stranded DNA | |
| DTT (10 mM) | 1.9 µL | Final concentration 1 mM; stabilizes enzyme activity and prevents oxidation |
- Add the components listed above to the reaction tube containing the digested circular dsDNA from the previous step.
- Mix thoroughly and ensure the reaction components are evenly distributed.
- Incubate at 25°C overnight to allow complete degradation of both linear dsDNA and ssDNA fragments.
Explanation: This step leaves only circular DNA intact, ensuring that no linear DNA contaminants interfere with downstream applications. Circular single-stranded DNA (ssDNA) remains protected from exonuclease activity due to its structure.
Bead Washing and Elution
Do Bead washing to remove any residual contaminants and purifies the remaining DNA. In all bead washing steps performed in this protocol, 1.5X ratio of beads relative to the volume of the DNA solution was used to ensure efficient purification and recovery of DNA.
| Procedure | Details | |
| Bead Washing and Elution | Use magnetic beads to wash the sample, eluting the DNA in 85 µL. | |
| Qubit and PCR Validation | Use 1 µL of the eluted DNA to measure concentration and confirm digestion of linear GRBV DNA. | |
| Outcome | in our experiment, from 22 µg total DNA, approximately 20 µg linear DNA was digested, leaving ~2 µg for the next step. |
Point: This step is very important. Any residual enzymes (e.g., exonucleases or restriction enzymes) can degrade circular DNA during subsequent steps. Perform this step carefully to ensure complete removal of enzymes and buffers.
Synthesis of Complementary Strand of circular single-stranded DNAs (ssDNA)
This step synthesizes the complementary strand for circular single-stranded DNA (ssDNA), effectively creating double-stranded DNA (dsDNA).
| Component | Volume | Details | |
| Circular ssDNA (Digested DNA) | 78 µL | Purified DNA from the previous step (2 µg) | |
| E. coli DNA Ligase I Buffer (10X) | 10 µL | Provides optimal conditions for ligase activity and strand synthesis | |
| dNTP (10 mM) | 4 µL | Supplies nucleotides for DNA synthesis | |
| Klenow DNA Polymerase I | 4 µL | Synthesizes the complementary strand, creating double-stranded DNA | |
| E. coli DNA Ligase | 4 µL | Facilitates ligation of nicked dsDNA | |
| DNase-free water | Adjust to 100 µL | Final reaction volume |
Set Up Reaction:
- Combine the above components in a clean microcentrifuge tube.
- Ensure the DNA, buffer, and enzymes are mixed gently to avoid damage to the DNA.
- Incubate the reaction at 16°C for 2.5 hour.
Point: During this incubation, Klenow DNA Polymerase I synthesizes the complementary strand of the circular ssDNA. E. coli DNA Ligase seals the nicks to form complete double-stranded DNA.
Bead Washing and Elution:
Use magnetic beads to purify the synthesized dsDNA.
Elute in an appropriate volume of DNase-free water (typically 50 µL).
Final Digestion with the restriction enzyme (KpnI)
This step was performed to produce linear double-stranded DNA from the GRBV genome. One strand of the resulting DNA represents the native strand of the circular ssDNA, while the other strand is the complementary sequence synthesized during the Klenow reaction. The digestion by KpnI ensures the DNA is linearized, which is necessary because the Nanopore library preparation kits require linear double-stranded DNA as input material for sequencing.
| Component | Volume | Details | |
| Circular dsDNA | ~ | Purified circular DNA from previous step | |
| KpnI-HF | 3 µL | 60 units | |
| 10X NEBuffer | 5 µL | Provides optimal conditions for KpnI activity | |
| DNase-free water | Adjust to 50 µL | Final reaction mixture |
- Combine the components in a clean microcentrifuge tube.
- Incubate at 37°C for 1 hour to allow digestion by KpnI.
- Final Bead Washing (Critical Step):
- Use magnetic beads to wash the reaction mixture thoroughly, ensuring all residual enzymes and buffer components are removed.
- Elute the purified linear DNA for further analysis.
Point:
We used KpnI because it is the one of restriction enzymes that cuts the GRBV genome at a single site, enabling precise linearization and having the full length of GRBV. Users of this protocol should select a restriction enzyme that cuts their viral genome at one specific site to achieve accurate linearization for downstream library preparation and sequencing.
- Measure DNA Concentration
Use Qubit to measure DNA concentration; in our case yield was ~4 µg.