AFLP Assays
Amplified Fragment Length Polymorphism (AFLP) is a versatile and sensitive technique for genome fingerprinting and genetic diversity analysis. Its widespread use stems from its ability to detect polymorphisms without prior sequence data. However, the precision and reproducibility of AFLP depend heavily on adhering to rigorous protocols.
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| AFLP assays detect genetic variation by digesting DNA with restriction enzymes, ligating adapters, amplifying selective fragments, and analyzing patterns—useful in genetics, forensics, and biodiversity studies. |
Essential Preparations for AFLP Assays
General Guidelines
- Always work on ice unless specified otherwise.
- Allow chemicals (except enzymes) to thaw completely and vortex briefly before use.
- Use ultrapure water (ddH₂O) and ensure consistency by using the same batch of chemicals across samples.
DNA Template Preparation
High-quality DNA is critical for AFLP success.
Quantification of DNA:
- Start with approximately 500 ng of genomic DNA in 5.5 µL of ddH₂O.
- If DNA concentration is insufficient, concentrate using a vacuum oven at 60°C or dilute with 1× TE buffer.
Handling Precautions:
- Perform DNA quantification at room temperature.
- Avoid contamination by using sterile tools and reagents.
Restriction-Ligation (RL) Reaction
The restriction-ligation step prepares the DNA fragments for amplification by attaching adaptors to restriction enzyme cut sites.
Required Chemicals and Volumes
Prepare a master mix for all samples based on the quantities in Table 1:
| Chemical | µL per Sample | Comments |
|---|---|---|
| 10× T4 Ligase Buffer | 1.1 | Vortex before use |
| 0.5 M NaCl | 1.1 | |
| 1 mg/mL BSA | 0.55 | Freshly diluted |
| MseI Adaptor Pair (50 µM) | 1 | Annealed prior to use |
| EcoRI Adaptor Pair (5 µM) | 1 | Annealed prior to use |
| MseI Restriction Enzyme | 1 U/sample | Thermo-sensitive |
| EcoRI Restriction Enzyme | 5 U/sample | Thermo-sensitive |
| T4 DNA Ligase | 0.6 U/sample | Thermo-sensitive |
| ddH₂O | Adjust to 5.5 µL | Ensure correct volume |
Protocol Steps
- Heat MseI and EcoRI adaptor pairs to 95°C for 5 minutes to denature. Cool gradually to room temperature and spin briefly.
- Combine ddH₂O, buffer, NaCl, BSA, adaptors, and enzymes to create a master mix.
- Aliquot 5.5 µL of the master mix into individual tubes.
- Add 5.5 µL of DNA (containing 500 ng) to each tube for a final reaction volume of 11 µL.
- Incubate at 37°C for 3 hours, followed by overnight incubation at 17°C.
Quality Check
- Run 5 µL of the RL product on a 1.5% agarose gel with ethidium bromide or GelRed.
- A successful RL reaction appears as a smear with brighter bands between 100–1,500 base pairs.
Preselective PCR Amplification
Preselective PCR narrows the pool of fragments while maintaining diversity for selective amplification.
Master Mix Composition
Prepare a master mix for preselective amplification as per Table 2:
| Chemical | µL per Sample |
|---|---|
| ddH₂O | 5.86 |
| 10× Taq Buffer | 1.14 |
| Primers (5 µM each) | 0.58 |
| dNTPs (10 mM) | 0.22 |
| RedTaq Polymerase | 0.2 |
Protocol Steps
- Add 2 µL of diluted RL product to 8 µL of the master mix for a total reaction volume of 10 µL.
- Run the thermal cycler:
- 72°C for 2 minutes.
- 20 cycles of 94°C (1 second), 56°C (30 seconds), 72°C (2 minutes).
- Final hold at 60°C for 30 minutes.
- Check the product by running 5 µL on a 1.5% agarose gel.
Storage
- Dilute preselective reactions 20-fold with 1× TE buffer.
- Store at 4°C for 1 day or at -20°C for long-term storage.
Selective PCR Amplification
Selective amplification enhances resolution by targeting specific fragments.
Master Mix Composition
Prepare a master mix similar to preselective PCR but adjust primers as per Table 2.
Protocol Steps
- Add 2 µL of diluted preselective product to 8 µL of the selective PCR master mix.
- Thermal cycler program:
- 94°C for 2 minutes.
- 9 cycles of 94°C (1 second), 65°C (reduce by 1°C per cycle for 30 seconds), 72°C (2 minutes).
- 23 cycles of 94°C (1 second), 56°C (30 seconds), 72°C (2 minutes).
- Final hold at 60°C for 30 minutes.
- Store at -20°C or proceed to analysis immediately.
Fragment Separation and Analysis
Sephadex Filtration:
- Add 200 µL of Sephadex solution to Multi-Screen plates and spin at 600×g. Repeat twice.
- Apply selective PCR products and spin again to collect the filtered product.
Sequencer Loading:
- Mix 9.8 µL Hi-Di formamide with 0.2 µL GeneScan ROX ladder per sample.
- Add 1.2 µL of filtered selective products.
- Heat at 95°C for 5 minutes and cool on ice.
- Load the samples onto the sequencer.
By adhering to the described protocols, researchers can achieve precise and reproducible results with AFLP. This comprehensive guide ensures the accuracy of each step, from restriction-ligation to fragment analysis, paving the way for breakthroughs in genetic and epigenetic studies. AFLP remains a robust tool, bridging molecular genetics and evolutionary research.