What is AFLP? The Complete Principle and Operation Process

Introduction

• 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.
• However, 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 to 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 Reagents	Description
Taq enzyme	DNA polymerase
EcoRI/MseI adapters	Restriction enzyme adapters
E+A primers	Primers for PCR amplification
M+C primers	Primers for PCR amplification
T4 DNA Ligase	DNA ligase enzyme
E and M oligonucleotides	Oligonucleotides for PCR
Agarose	Gel matrix for electrophoresis
Ammonium persulfate	Catalyst for gel casting and DNA denaturation
Acrylamide	Component of gel matrix for high-resolution sequencing
Urea	Denaturing agent for electrophoresis
Silver nitrate	Stain for visualizing DNA bands
Formamide	Denaturing agent for DNA sequencing gels
dNTPs	Deoxynucleotide triphosphates for PCR
Xylene cyanol	Tracking dye for electrophoresis
Acetic acid	Used in gel preparation
Glass silane	Coating agent for glass plates in gel electrophoresis
50bp Marker	DNA marker of 50 base pairs for size reference

Experimental Procedure

1. Genomic DNA Extraction and Purification

Refer to DNA extraction methods.

DNA Purification:

• 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.
• Add 1/10 volume of NaAc and twice the volume of pre-cooled absolute ethanol, and place at -20°C for at least 2 hours. Centrifuge at 10,000g for 10 minutes.
• Wash the DNA precipitate twice with 70% ethanol, 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.

Note:

• 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, add:

• 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 minutes. 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:

• Initial denaturation at 94°C for 90 seconds,
• Denaturation at 94°C for 30 seconds,
• Annealing at 56°C for 1 minute,
• Extension at 72°C for 1 minute,
• Repeat the above steps for 30 cycles,
• Final extension at 72°C for 10 minutes (using a PCR machine from PE company).

After the reaction, 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:

• Initial denaturation at 94°C for 90 seconds,
• Denaturation at 94°C for 30 seconds,
• Annealing at 65°C for 1 minute, then perform 13 cycles (decrease the temperature by 0.7°C per cycle),
• Denaturation at 94°C for 30 seconds,
• Annealing at 56°C for 1 minute,
• Extension at 72°C for 1 minute, then perform 25 cycles,
• Final extension at 72°C for 5 minutes (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 minutes (using a PCR machine from PE company). After denaturation, 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, then add 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. After electrophoresis, 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 minutes. Reserve the fixation solution after fixation.
3. Rinsing: Wash the gel three times with deionized water for 2 minutes each time.
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 minutes. After rinsing the gel with deionized water for 10 seconds, 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.

Summary

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