The AFLP (Amplified Fragment Length Polymorphism) technique is used to visualize hundreds of amplified DNA bands of restriction fragments simultaneously (Vos et al., 1995).  The AFLP band patterns, or fingerprints, can be used for many purposes, such as monitoring the identity of an individual/population or the degree of similarity among individuals/populations. Polymorphisms in band patterns map to specific loci, allowing the individuals to be genotyped or differentiated based on the alleles they carry.   This technique combines the power of restriction fragment length polymorphism (RFLP) with the flexibility of PCR-based technology by ligating primer-recognition sequences (adaptors) to the restricted DNA.

Protocol (Using AFLP Analysis Kit I- Life technologies)

Add the following  to a 1.5ml microcentrifuge tube

Mix gently and collect the reaction by brief centrifugation. Incubate the mixture for 2 hours at 37º C.

Incubate the mixture for 15 minutes at 70º C to inactivate the restriction endonucleases. Place tube on ice and collect contents by brief centrifugation.

Ligation of adapters

Add the following to the digested DNA from step 3 of Section 5.1

Adapter ligation solution        12 µl

T4 DNA ligase                      0.5 µl

Mix gently at room temperature, centrifuge briefly to collect contents, and incubate at 20¡ãC ¡À 2¡ãC for 2 h.

Perform 110 dilution of the ligation mixture as follows

a. Take 1 µl of the reaction mixture and transfer to a 1.5-ml microcentrifuge  tube.

b. Add 9 µl TE buffer and mix well.

The unused portion of the reaction mixture may be stored at ‡°20¡ãC.

Restriction digestion of genomic DNA 

Pre amplification Reactions

 Add the following to a 0.2- or 0.5-ml thin-walled microcentrifuge tube

Mix gently and centrifuge briefly to collect reaction.

Selective AFLP Amplification

For each primer pair, add the following components to a 1.5-ml microcentrifuge tube and label it “Mix 1”

Add the following components to another 1.5-ml microcentrifuge tube and label it “Mix 2”.

Mix gently and centrifuge briefly to collect reaction.

• Perform one cycle at 94¡ãC for 30 s; 65¡ãC for 30 s; and 72¡ãC for 60 s.

• Lower the annealing temperature each cycle 0.7¡ãC during 12 cycles. This gives a touch down phase of 13 cycles.

Perform 23 cycles at 94¡ãC for 30 s; 56¡ãC for 30 s; and 72¡ãC for 60 s.

Gel Analysis

• After PCR, add an equal volume (20 µl) of formamide dye (98% formamide, 10 mM EDTA, place on bromophenol blue, xylene cyanol) to each reaction. Heat thesamples for 3 min at 90¡ãC and immediately ice.
• Pour a 5% polyacrylamide gel with 0.4-mm spacers and sharkstooth combs.
• Pre-electrophorese the gel at constant power (~55 W) for ~20 min.
• Load 3 µl of each sample on the gel.
• Electrophorese at constant power until xylene cyanol (slower dye) is two-thirds down the length of the   gel.
• Silver stain the gel and score the bands.

Applications

• Establishing linkage groups in crosses
• Assessing the degree of relatedness or variability among cultivars
• A genetic map of markers showing Mendelian inheritance can be constructed from  AFLP data