AFLP

As AFLP (Abbr. ENGL. A mplified f ragment- l ength p olymorphism ) in which is molecular biology , a technique referred to, with which a genetic fingerprint can be created. In AFLP, the DNA is cut into fragments by two restriction enzymes . Then some fragments are duplicated ( amplified ) with the help of two polymerase chain reactions . Differences in the number of restriction sites result in fragments of different lengths, the pattern of which can be used on an electrophoresis gel to differentiate between individuals and also to show close relationships.

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

Steps of the AFLP

Restriction

The AFLP technique, like the RFLP or ARDRA, uses the fact that there are very many sites in the genome that are cut by restriction enzymes. By mutations new ones are added, or it go which lost. Restriction enzymes (only those of type II are used) cut only at certain points in the genome that contain a so-called recognition sequence. The sequences used are 4 and 6, rarely 8 base pairs long. Since the genome is very large, chance alone ensures that these locations are distributed in high numbers in the genome.

After the DNA has been purified, it is cut up with the aid of two restriction enzymes. For this purpose, one usually uses an enzyme whose recognition sequence is quite short with four base pairs and thus carries out frequent cuts ( frequent cutter ), such as B. Mse I. The other enzyme usually has a recognition sequence of six, less often eight base pairs, so cuts less often (called “ rare cutter ”), e.g. B. Eco RI . This results in three types of fragments: Fragments with both ends of restriction enzyme 1 ( Mse I- Mse I), those with both ends of enzyme 2 ( Eco RI- Eco RI) and hybrids ( Mse I- Eco RI).

Ligation

Restriction enzymes used in AFLP cut the DNA double strand with sticky ends. This means that one strand protrudes as a single strand with a short piece. A reaction ( called ligation ) now takes place in which adapters are attached to these ends. These consist of the corresponding counterpart to the protruding end of the restriction fragments and a 10-15 base pair double strand with a known sequence. The fragments now consist of a core consisting of the restriction fragment and two surrounding adapters. The ends of the adapters corresponding to the restriction sites usually do not correspond to the actual restriction sites, but are changed in a base. This allows restriction and ligation to take place in one reaction step without the restriction enzymes removing the ligated adapters again.

First amplification

If you cut a genome with restriction enzymes, thousands of fragments are created that are quite difficult to evaluate. In AFLP, a part of the fragments is now specifically duplicated (amplified) using a polymerase chain reaction (PCR). In this case, two single-stranded DNA oligonucleotides are added, which attach to the corresponding counter-sequences in the DNA. The oligonucleotides serve as a starting point ( primer ) for an amplification reaction of the DNA (polymerisation). The opposite strand is used as a template to extend the primer to the complementary single strand. This doubles the number of DNA strands with each step.

In AFLP, the counter-sequences of the adapters are used as primers. Since the sequences in the restriction fragments themselves are not known, the AFLP technique creates the primer attachment points through the adapter itself. However, the primer does not correspond 100% to the adapter sequence. It comprises part of the adapter, the cutting sequence and, in the first amplification step, one or two additional bases within the fragment. With this base (s), not all fragments are duplicated, but only a part. As a result, these bases select fragments, which is why we also speak of selective bases . If the primers contain as a selective base at the 3 'end z. B. a cytosine , only those fragments are amplified that contain a guanine at the corresponding point (all fragments that contain an adenine , thymine or cytosine at this point are not amplified). With a selective base per primer only 1/4 × 1/4 = 1/16 of the fragments is amplified, with two selective bases per primer only (1/4) ⁴ = 1/256. This step results in a drastic reduction in fragments.

Second amplification

Since the number of fragments is still very high, a further amplification step is added. In this, however, mostly three and sometimes four selective bases are used, so that there is a further reduction in the number of fragments. The special feature of this step is that the primers for the adapter of the rare interface must have a detectable property; one also speaks of labeled primers. They can be radioactive or have a fluorescent group attached. Since there are three types of fragments, depending on the restriction site ( see above ), there are now three possibilities. Fragments with two cleavage sites or adapters of the common restriction enzyme (e.g. Mse I- Mse I) are not marked, as a result of which they fall out of the investigation. Fragments with two rare restriction sites ( rare cutter , z. B. Eco RI- Eco RI) and hybrids that are each labeled, are detected. The probability that there is a frequent cutter position between two interfaces of the rare cutter is very high, so that Eco RI- Eco RI rarely occur. Furthermore, the frequent cutter adapters are selected in such a way that fragments with two of these adapters form a hairpin structure and the amplification is thus inhibited. As a result, especially hybrids ( Eco RI- Mse I) are reproduced, so that double markings of a single fragment play practically no role.

After the second amplification, the labeled fragments are separated using electrophoresis . This can e.g. B. happen in a polyacrylamide gel ( polyacrylamide gel electrophoresis ). The individual fragments are then made visible with a photo film (with radioactive labeling) or with the help of a laser and a color detector (with fluorescent labeling).

The analysis usually works towards about 50 to 120 fragments per primer combination, since this number offers good statistical support.

application

AFLP is a fairly simple method and, compared to satellite markers, quite cheap. Primers do not need a long development time, which is why AFLP has become very important in studies of population structures . Due to the large number of markers , very closely related species can also be phylogenetically differentiated. The AFLP is therefore often used when groups of species can not be analyzed or can only be analyzed very poorly using DNA sequences .

In principle, the AFLP can also be used for genetic fingerprints in criminology, but there have already been developed corresponding standards with mini-satellites .

Individual evidence

1. ↑ Vos, P. et al. (1995): AFLP: a new technique for DNA fingerprinting. In: Nucleic Acids Res. 23 (21): 4407-4414. PMID 7501463 , PMC 307397 (free full text).