Amplified fragment length polymorphism

As AFLP ( Abbreviation for. Amplified fragment - length polymorphism ) is in molecular biology refers to a technique by which a genetic fingerprint can be created. In the AFLP DNA is cut by two restriction enzymes into fragments. Thereafter, with the help of two polymerase chain reactions some fragments duplicated ( amplified ). By differences in the number of restriction sites created different lengths of fragments, which pattern may be used on an electrophoresis gel for distinguishing individuals, and also for displaying the near affinities.

The AFLP technique was developed in 1995 by the Working Group of Pieter Vos.

Steps of the AFLP

Restriction

Using AFLP technique, as well as the RFLP, the fact that many sites are present in the genome, which are cut by restriction enzymes. Mutations new added or lost any. Restriction enzymes ( only the Type II are used) cut only at specific sites in the genome that contain a so-called recognition sequence. The sequences 4 and 6 are used, often 8 base pairs long. Since the genome is very large, is guaranteed only by the coincidence that these sites are present in high numbers distributed in the genome.

After purification, the DNA is cut with the help of these two restriction enzymes. For this use is usually an enzyme whose recognition sequence is quite short with four base pairs and thus frequent cuts performs ( frequent cutter ), such as MseI. The other enzyme usually has a recognition sequence of six base pairs of rare eight, so rarely cuts ( "rare cutter" referred to ), for example EcoRl. This results in three types of fragments: fragments with both cut ends of the restriction enzyme 1 ( MseI - MseI ) connected to both ends of the enzyme 2 (EcoRI - EcoRI) and Hybrid ( MseI - EcoRI).

Ligation

In the AFLP used restriction enzymes cut the DNA double strand with protruding (English sticky ) ends. That is, one strand is produced with a short piece of a single strand. Now, a reaction ( called ligation ) are mounted in the adapter to these ends is done. These consist of the corresponding counterpart to the projecting end of the restriction fragments and a 10-15 base pair long double-strand of known sequence. The fragments consist now of a core consisting of the restriction fragment and the surrounding two adapters. The corresponding restriction sites ends of the adapter does not usually correspond to the actual restriction sites, but have changed in a base. Can be made by restriction and ligation, in a reaction step, without the restriction enzymes to remove the adapter ligated again.

First amplification

When we cut DNA with restriction enzymes, caused thousands of fragments that are quite difficult to evaluate. Reproduced at the AFLP ( amplified ) is now a part of the target fragments with a polymerase chain reaction (PCR). In this two single-stranded DNA oligonucleotides are added, which bind to the equivalent sequences in the DNA. The oligonucleotides serve as a starting point ( primer) for amplification reaction of DNA ( polymerization). To this end, the opposite strand is used as a template to extend the primer. Characterized the number of the DNA strands is doubled for each step.

In the AFLP one now uses the opposite sequences of the adapter as a primer. Since you do not know the sequences in the restriction fragments themselves, the AFLP technique, the primer annealing points creates through the adapter itself, the primer is not up to 100% of the adapter sequence. It comprises a portion of the adapter, the cutting sequence, and in the first amplification step, one or two bases in the fragment. Through this base (s ) are not reproduced all the fragments, but only a part. Thus, these bases select fragments, which is why it is also called selective bases. Containing primers as a selective base at the 3'-end, for example, a cytosine, only those fragments are amplified, which at the corresponding point, a guanine containing ( all fragments that contain at this point is an adenine, thymine, or cytosine, not amplified ). At a selective base per primer only 1/4 × 1 /4 = 1/16 of the fragments amplified in two selective bases per primer even only ( 1/4) 4 = 1 / 256th By this step, there is a drastic reduction of the fragments.

Second amplification

As the number of fragments is still very high is followed by a further amplification step. In this, however, are usually three, sometimes four selective bases used so that there is again a reduction in the number of fragments. The special thing in this step is that the primers for the adapter of the rare interface must have a detectable property, one also speaks of labeled primers. You can be radioactive or contain a fluorescent group attached. Since there are three types of fragments, depending on the restriction site (see above), now three possibilities arise again. Fragments with two interfaces or adapters of the frequent restriction enzyme (eg MseI MseI ) are not marked, making them drop out of the study. Fragments with two rare restriction sites (rare cutter, such as Eco RI -Eco RI ), and hybrids of each labeled are detected. The probability that there is a frequent cutter- point between two interfaces of the rare cutters, is very high, so that EcoRI - EcoRI are rare. Furthermore, the frequent cutter adapter selected so that two of these fragments with adapter forming a hairpin structure, and the amplification is thus inhibited. Consequently, especially hybrids ( EcoRI - MseI ) are duplicated, so double labeling of a single fragment play virtually no role.

After the second amplification, the labeled fragments are separated by means of electrophoresis. This can be done for example in a polyacrylamide gel ( polyacrylamide gel electrophoresis ). The individual fragments are then taken with a photographic film (in case of radioactive labeling ) or by means of a laser and a color detector (for fluorescent label ) visible.

Usually worked in the analysis to about 50 to 120 fragments per primer combination, as this guarantees a good statistical support.

Application

AFLP is a fairly simple and compared to markers rather favorable method. It does not require long development time of primers, whereby the AFLP has gained great importance in studies of population structures. Also can be due to the high number of markers also very closely related species differ phylogenetically. So AFLP is often used when can not or only very poorly analyze groups of species by DNA sequences.

The AFLP can in principle also be used for genetic fingerprinting in forensics, however, there has already developed such standards with mini-satellites.