Different organisms have specific sequences that are particular to only one type of oraganism or that help us differentiate among organisms. Some example of such repeats are VNTRs(Variable number tandem repeats), SNPs( single nucleotide profiling).These sequence help us create a genome profile for organisms and helps us to group organisms or humans based on the type of genome profile. Among many others, AFLP(Amplified fragment length polymorphism) is one such technique that helps us identify such repeats.
WHAT IS AFLP?
AFLP or Amplified Fragment Length Polymorphism is a PCR based tool used in molecular diagnostic techniques. It was developed in the early 1990s by Keygene. AFLP are DNA fragment obtained after digestion by restriction enzymes . The ligation products are then digested to oligonucleotide adapters followed by selective amplification using PCR. This is the reason, AFLP technique is known to be a combination of RFLP and PCR.
Selective Fragment Length Amplification (SFLA) and Selective Restriction Fragment Amplification (SRFA) are synonyms sometimes used to refer to AFLPs. The PCR primers consist of core sequences that are part of the adapters, restriction enzyme specific sequence and 1-5 selective nucleotides. The higher the number of selective nucleotides lower will be the number of bands obtained.
The AFLP technology has the capability to detect various polymorphisms in different genomic regions simultaneously. It is also highly sensitive and reproducible. As a result, AFLP has become widely used for the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria. The AFLP technology has been used in criminal and paternity tests, also to determine slight differences within populations, and in linkage studies to generate maps for quantitative trait locus (QTL) analysis.
The technique was originally described by Vos and Zabeau in 1993.In detail, the procedure of this technique is divided into three steps:
- Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments.
- Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.
- Electrophoretic separation of amplicons on a gel matrix, followed by visualization of the band pattern.
- RESTRICTION DIGESTION
At first the sample of DNA are cut using restriction enzymes. Depending on the number of restriction sites in the DNA samples restriction fragment are produced . For example, if a sample 1 has DNA with one restriction site for EcoR1 then it will produce only 2 fragment, and if sample 2 has DNA with restriction sites for EcoR1 then it produces 3 fragment.
2. ADAPTOR ADDITION AND SELECTIVE AMPLIFICATION
Once the restriction fragments are generated the next step involves amplification of these fragments. But if we directly go for PCR the reaction may face a problem as the restriction digestion creates sticky ended restriction fragments. So, during PCR there is probability that the primers may bind to the sticky ends. To avoid this, to the DNA fragments adaptors are added which convert the sticky end to blunt ended fragments.
In the next steps PCR amplification is performed and primers with core sequences that are part of the adaptors are added.
Preamplification- This first amplification that uses the adaptor sequences as primers allows only those fragments to be amplified that have adaptors ligated to their extremeties. Additionally to adaptor sequences, the primers used for pre-selective amplification also have an supplementary base. This extra base allows another selection that amplifies 1/4 th of the fragment that have adaptors ligated at the extremeties. Till here the DNA can be visualized on agarose gel.
Selective amplification- The aim of this step is to restrict the level of polymorphism and to label the DNA. For this second amplification 3 more nucleotides are added at 3′ end of the primer sequence that is used in preamplification. These additional nucelotides make amplification more selective and decrease the number of restriction fragments amplified. Also, in this step one of the primers is labelled with florescent dye that helps in visualizing the DNA during migration. The PCR products are visualized using acrylamide gel electrophoresis.
Once the PCR is completed the amplified fragment are run on polyacrylamide gel and DNA are separated based on their charge and mass. The bands are then visualized and different band samples compared and studied for polymorphism.