What is AFLP? The Complete Principle and Operation Process

Introducción

• AFLP is a DNA molecular marker technology that detects DNA polymorphism by restricting the lengths of fragments produced by restriction endonucleases.
• The main characteristics of the AFLP technique are:
  • Requires a small amount of DNA for analysis, only 0.5g.
  • Exhibits good reproducibility.
  • Demonstrates strong polymorphism.
  • Offers high resolution.
  • Does not require Southern hybridization.
  • Suitable for a wide range of samples.
  • Exhibits stable heredity.
• In terms of technical features, AFLP is a product combining RAPD and RFLP.
• It overcomes the disadvantages of the complexity and radioactive hazards of RFLP technology, and the instability and dominance of genetic markers in RAPD while incorporating the strengths of both.
• In recent years, this technology has been continuously improved and developed, making AFLP rapidly become the most effective molecular method to date.

The Principle of AFLP

• AFLP is also a DNA molecular marker technology that detects DNA polymorphism by restricting the lengths of fragments produced by restriction endonucleases.
• Sin embargo, AFLP involves amplifying the enzyme-cut fragments through PCR reaction first, and then electrophoresing the amplified enzyme-cut fragments on a high-resolution sequencing gel. Polymorphism is detected based on the different lengths of the amplified fragments.
• In the experiment, the enzyme-cut fragments are first ligated to artificial adapters containing compatible cohesive ends. The sequence of these cohesive ends, along with the adapter sequence, serves as the binding site for subsequent PCR reactions.
• Depending on the requirements, different primers with 1 a 3 selective nucleotides added at the ends can be used to achieve selective amplification. These selective nucleotides enable the primers to selectively recognize enzyme-cut fragments with specific pairing sequences, leading to specific amplification.

Reagent Used in the Test

Experimental Procedure

1. Genomic DNA Extraction and Purification

Refer to DNA extraction methods.

Purificación de ADN:

• Electrophoresis of DNA fragments on a 0.8% agarose gel (containing 0.5μg/ml ethidium bromide, EB), take out 1/3 of the extracted genomic DNA for purification.
• Firstly, replenish the volume of the extracted DNA with TE buffer to a total volume of 50 µl.
• Extract once with phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). Centrifuge and transfer the supernatant to an Eppendorf tube.
• Agregar 1/10 volume of NaAc and twice the volume of pre-cooled absolute ethanol, and place at -20°C for at least 2 horas. Centrifuge at 10,000g for 10 minutos.
• Wash the DNA precipitate twice with 70% etanol, air dry, and dissolve in 30 μl TE buffer.
• Measure A260 and A280 values using a UV-2401PC (Shimadzu) UV spectrophotometer for quantification.
• Electrophoresis of DNA fragments on a 0.8% agarose gel (containing 0.5μg/ml EB) for size determination.

Nota:

• For 0.1-0.2g tissue, dissolve in 100μl solution E. For 0.5g tissue, increase solution E to 300μl. At this point, the DNA concentration is approximately 100ng/μl.

2. Restriction Enzyme Digestion and Ligation

In a 0.2ml centrifuge tube, agregar:

• Approximately 250ng template DNA,
• 2.5μl 10× restriction enzyme digestion buffer,
• 2.5μl 10× T4 DNA ligase buffer,
• 5 units of EcoRI,
• 5 units of MseI,
• 2 units of T4 DNA ligase,
• 50pmol MseI adapter,
• Double distilled water to a total volume of 25μl.

Incubate the mixture in a PCR thermocycler set at 37°C overnight. After digestion, inactivate the enzymes by incubating at 65°C for 20 minutos. Store the reaction at -20°C for further use as a pre-amplification template.

3. Pre-amplification

Take 3μl of the enzyme-digested and ligated product and add:

• 75ng of E+A primer,
• 75ng of M+C primer,
• 15mM Mg2+,
• 25mM dNTPs,
• 1 unit of Taq polymerase,
• 3μl of 10× PCR buffer,
• Add double distilled water to a total volume of 30μl.

Set the PCR reaction parameters as follows:

• Desnaturalización inicial a 94°C para 90 segundos,
• Denaturation at 94°C for 30 segundos,
• Annealing at 56°C for 1 minuto,
• Extension at 72°C for 1 minuto,
• Repeat the above steps for 30 ciclos,
• Extensión final a 72°C para 10 minutos (using a máquina de PCR from PE company).

Después de la reacción, analyze the amplification products by electrophoresis on a 0.8% agarose gel (containing 0.5μg/ml EB). Take 3μl of the product and dilute it 50 times for further use as a template for selective amplification.

4. Selective PCR Amplification

Take 3μl of the diluted product and add:

• 75ng of EcoRⅠ selective primer,
• 75ng of MseⅠ selective primer,
• 15mM Mg2+,
• 25mM dNTPs,
• 1 unit of Taq polymerase,
• 3μl of 10× PCR buffer,
• Add double distilled water to a total volume of 30μl.

Set the PCR reaction parameters as follows:

• Desnaturalización inicial a 94°C para 90 segundos,
• Denaturation at 94°C for 30 segundos,
• Annealing at 65°C for 1 minuto, then perform 13 ciclos (decrease the temperature by 0.7°C per cycle),
• Denaturation at 94°C for 30 segundos,
• Annealing at 56°C for 1 minuto,
• Extension at 72°C for 1 minuto, then perform 25 ciclos,
• Extensión final a 72°C para 5 minutos (using a PCR machine from PE company).

First, analyze the products of selective amplification by electrophoresis on a 0.8% agarose gel (containing 0.5μg/ml EB).

5. Gel Electrophoresis

Separate the amplified products using a 6% denaturing polyacrylamide gel (0.5mm thickness) and 1× TBE electrophoresis buffer (BIO-RAD sequencing electrophoresis apparatus).

• Remove the comb and pre-run the gel at a constant power of 140W for 30 minutes until the temperature reaches 47-49°C. Make sure to wash out urea from each well.
• For the selective amplification products, add an equal volume of loading buffer (98% formamide, 10mM EDTA, 0.25% xylene cyanol, 0.25% bromophenol blue) and denature at 94°C for 5 minutos (using a PCR machine from PE company). Después de la desnaturalización, quickly place on ice until loading.
• Load 8μl of each sample into each lane. Initially, run the gel at a constant power of 100W for about 2 minutes to quickly concentrate the samples at the bottom of the wells. Then adjust to a constant power of 60W, keeping the temperature around 43°C, until the bromophenol blue reaches about 2/3 of the distance from the top of the gel to the glass plate. End the electrophoresis.

6. Silver Staining

• Fixation Solution: Add 100ml of glacial acetic acid to 900ml of deionized water or double-distilled water.
• Staining Solution: Dissolve 1g AgNO3 in 1L of deionized water, Luego añade 1.5 ml of 37% formaldehyde.
• Developing Solution: Dissolve 30g Na2CO3, 1.5 ml of 37% formaldehyde, and 2mg of sodium thiosulfate in 1L of deionized water.

1. Después de la electroforesis, place the glass plate with the gel into a plastic tray for silver staining.
2. Fixation: Add the fixation solution and gently shake on a shaker for 30 minutos. Reserve the fixation solution after fixation.
3. Rinsing: Wash the gel three times with deionized water for 2 minutos cada vez.
4. Staining: Place the gel into a staining tray and pour in the staining solution (kept at 4°C). Gently shake on a shaker for 30 minutos. After rinsing the gel with deionized water for 10 segundos, transfer it to the developing tray.
5. Development: Add the developing solution (kept at 4°C) and gently shake until the bands stop increasing.
6. Termination: Add the used fixation solution from step 2 and agitate for a few minutes. Rinse with distilled water for several minutes once optimal results are achieved.
7. After removing water droplets from the gel and glass plate, place it on a lightbox and photographed using a Nikon digital camera.

Resumen

Thank you for providing the complete AFLP experimental procedure! If you have any questions or need further assistance, please feel free to ask us. We’re happy to provide support and answers.