Amplified fragment length polymorphism

Amplified Fragment Length Polymorphism

Amplified Fragment Length Polymorphism (AFLP) is a powerful DNA fingerprinting technique used in genetics and molecular biology to detect polymorphisms in DNA sequences. This method is widely used for various applications, including genetic mapping, population genetics, and the identification of genetic diversity within and between species.

Overview

AFLP is a PCR-based technique that involves the selective amplification of restriction fragments from a total digest of genomic DNA. The process is highly sensitive and can detect multiple polymorphisms simultaneously without prior knowledge of the DNA sequence.

Methodology

The AFLP technique involves several key steps:

1. DNA Extraction: Genomic DNA is extracted from the organism of interest.

2. Restriction Digestion: The DNA is digested with two restriction enzymes, typically a rare cutter and a frequent cutter, to generate a large number of DNA fragments.

3. Ligation of Adaptors: Short double-stranded adaptors are ligated to the sticky ends of the restriction fragments. These adaptors serve as primer binding sites for subsequent PCR amplification.

4. Selective Amplification: A subset of the restriction fragments is selectively amplified using PCR. This is achieved by using primers that anneal to the adaptor sequence and extend into the restriction fragment, with additional selective nucleotides at the 3' end.

5. Electrophoresis and Detection: The amplified fragments are separated by size using gel electrophoresis or capillary electrophoresis. The resulting pattern of bands, or "fingerprint," is visualized and analyzed.

Applications

AFLP is used in various fields of biological research:

- Genetic Mapping: AFLP markers are used to construct genetic linkage maps, which are essential for identifying genes associated with specific traits. - Biodiversity Studies: AFLP is employed to assess genetic diversity within and between populations, species, and ecosystems. - Phylogenetics: The technique helps in reconstructing evolutionary relationships among species. - Breeding Programs: AFLP markers assist in marker-assisted selection and breeding of plants and animals.

Advantages and Limitations

Advantages

- High Resolution: AFLP can detect a large number of polymorphisms across the genome. - No Prior Sequence Information Required: The technique does not require prior knowledge of the DNA sequence. - Reproducibility: AFLP is highly reproducible and can be used to compare genetic material from different sources.

Limitations

- Complexity: The technique involves multiple steps and requires careful optimization. - Dominant Markers: AFLP markers are typically dominant, which can complicate the interpretation of heterozygous loci.

Also see

- Restriction Fragment Length Polymorphism - Polymerase Chain Reaction - Genetic Mapping - DNA Fingerprinting