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Amplified fragment length polymorphism (AFLP)
This is a highly sensitive method for detecting polymorphism throughout the genome, and it is
becoming increasingly popular. It is essentially a combination of RFLP and RAPD methods, and
it is applicable universally and is highly reproducible. It is based on PCR amplification of
genomic restriction fragments generated by specific restriction enzymes and oligonucleotide
adapters of few nucleotide bases (Vos et al., 1995). It is a novel DNA fingerprinting technique.
DNA fingerprinting involves the display of a set of DNA fragments from a specific DNA sample.
Fingerprints are produced without prior sequence knowledge, using a limited set of genetic
primers. AFLP technique uses stringent reaction conditions for primer annealing and combines
the reliability of RFLP technique with the power of PCR technique.
In principle, AFLP involves the following steps:
1. DNA is cut with restriction enzymes (generally by two enzymes), and double-stranded (ds)
oligonucleotide adapters are ligated to the ends of the DNA fragments.
2. Selective amplification of sets of restriction fragments is usually carried out with ^P-labeled
primers designed according to the sequence of adaptors plus 1-3 additional nucleotides. Only
fragments containing the restriction site sequence plus the additional nucleotides will be
amplified.
3. Gel analysis of the amplified fragments.
The amplification products are separated on highly resolving sequencing gels and visualized
using autoradiography. Fluorescent or silver staining techniques can be used to visualize the
products in cases where radiolabeled nucleotides are not used in the PCR.
The restriction fragments for amplification are generated by two restriction enzymes, a
rare cutter and a frequent cutter. The figure shows that fragments are generated from EcoRI and
MseI. Oligonucleotides are generally used as adapters and primers. AFLP adapters consist of a
core sequence and an enzyme specific sequence.
The restriction fragments with their 5' protruding ends are obtained and then adapters are
ligated. The sequence of the adapters and the adjacent restriction site serves as primer-binding
sites for subsequent amplification of the restriction fragments. Selective nucleotides are included
at the 3' ends of PCR primers, which therefore can only prime DNA synthesis from a subset of
the restriction sites. Only those restriction fragments in which nucleotides flanking the restriction
site match the selective nucleotides will be amplified.
The AFLP procedure results in predominant amplification of restriction fragments having
a rare cutter sequence on one end and a frequent cutter sequence on the other end.
The method allows specific co-amplification of a high number of restriction fragments.
Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide
gels.
This method generates a large number of restriction fragment bands, thereby facilitating
the detection of polymorphism. Choosing the different base number and composition of
nucleotides in the adapters can control the number of DNA fragments that are amplified.
Although not many maps using AFLP markers have been developed so far, this method is now
widely used for developing polymorphic markers. AFLP is a technique that detects genomic
restriction fragments and resembles RFLP in this respect, with the major difference that PCR
amplification instead of Southern blotting is used for detection of restriction fragments. In
general, there is a linear correlation between the number of amplified fragments and genome size.
Most AFLP fragments correspond to unique positions on the genome and hence can be exploited
as landmarks in genetic and physical maps, each fragment being characterized by its size and its
primers required for amplification. A comparison of the properties of AFLPs in relation to other
markers has been shown in Table (1).
Advantages
1. This technique is extremely sensitive.
2. It has high reproducibility, rendering it superior to RAPD.
3. It has widescale applicability, proving extremely proficient in revealing diversity.
4. It discriminates heterozygotes from homozygotes when a gel scanner is used.
5. It permits detection of restriction fragments in any background or complexity, including pooled
DNA samples and cloned DNA segments.
6. It is not only a simple fingerprinting technique, but can also be used for mapping.
Disadvantages
1. It is highly expensive and requires more DNA than is needed in RAPD (1 mg per reaction).
2. It is technically more demanding than RAPDs, as it requires experience of sequencing gels.
3. AFLPs are expensive to generate as silver staining, fluorescent dye, or radioactivity detect the
bands.
Table 1. Comparative properties of different markers.

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AFLP.doc

  • 1. Amplified fragment length polymorphism (AFLP) This is a highly sensitive method for detecting polymorphism throughout the genome, and it is becoming increasingly popular. It is essentially a combination of RFLP and RAPD methods, and it is applicable universally and is highly reproducible. It is based on PCR amplification of genomic restriction fragments generated by specific restriction enzymes and oligonucleotide adapters of few nucleotide bases (Vos et al., 1995). It is a novel DNA fingerprinting technique. DNA fingerprinting involves the display of a set of DNA fragments from a specific DNA sample. Fingerprints are produced without prior sequence knowledge, using a limited set of genetic primers. AFLP technique uses stringent reaction conditions for primer annealing and combines the reliability of RFLP technique with the power of PCR technique. In principle, AFLP involves the following steps: 1. DNA is cut with restriction enzymes (generally by two enzymes), and double-stranded (ds) oligonucleotide adapters are ligated to the ends of the DNA fragments. 2. Selective amplification of sets of restriction fragments is usually carried out with ^P-labeled primers designed according to the sequence of adaptors plus 1-3 additional nucleotides. Only fragments containing the restriction site sequence plus the additional nucleotides will be amplified. 3. Gel analysis of the amplified fragments. The amplification products are separated on highly resolving sequencing gels and visualized using autoradiography. Fluorescent or silver staining techniques can be used to visualize the products in cases where radiolabeled nucleotides are not used in the PCR. The restriction fragments for amplification are generated by two restriction enzymes, a rare cutter and a frequent cutter. The figure shows that fragments are generated from EcoRI and MseI. Oligonucleotides are generally used as adapters and primers. AFLP adapters consist of a core sequence and an enzyme specific sequence. The restriction fragments with their 5' protruding ends are obtained and then adapters are ligated. The sequence of the adapters and the adjacent restriction site serves as primer-binding sites for subsequent amplification of the restriction fragments. Selective nucleotides are included at the 3' ends of PCR primers, which therefore can only prime DNA synthesis from a subset of the restriction sites. Only those restriction fragments in which nucleotides flanking the restriction site match the selective nucleotides will be amplified. The AFLP procedure results in predominant amplification of restriction fragments having a rare cutter sequence on one end and a frequent cutter sequence on the other end.
  • 2. The method allows specific co-amplification of a high number of restriction fragments. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. This method generates a large number of restriction fragment bands, thereby facilitating the detection of polymorphism. Choosing the different base number and composition of nucleotides in the adapters can control the number of DNA fragments that are amplified. Although not many maps using AFLP markers have been developed so far, this method is now widely used for developing polymorphic markers. AFLP is a technique that detects genomic restriction fragments and resembles RFLP in this respect, with the major difference that PCR amplification instead of Southern blotting is used for detection of restriction fragments. In general, there is a linear correlation between the number of amplified fragments and genome size. Most AFLP fragments correspond to unique positions on the genome and hence can be exploited as landmarks in genetic and physical maps, each fragment being characterized by its size and its primers required for amplification. A comparison of the properties of AFLPs in relation to other markers has been shown in Table (1). Advantages 1. This technique is extremely sensitive. 2. It has high reproducibility, rendering it superior to RAPD. 3. It has widescale applicability, proving extremely proficient in revealing diversity. 4. It discriminates heterozygotes from homozygotes when a gel scanner is used. 5. It permits detection of restriction fragments in any background or complexity, including pooled DNA samples and cloned DNA segments. 6. It is not only a simple fingerprinting technique, but can also be used for mapping. Disadvantages 1. It is highly expensive and requires more DNA than is needed in RAPD (1 mg per reaction). 2. It is technically more demanding than RAPDs, as it requires experience of sequencing gels. 3. AFLPs are expensive to generate as silver staining, fluorescent dye, or radioactivity detect the bands.
  • 3. Table 1. Comparative properties of different markers.