AFLP-PCR 

 AFLP is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice

of genetic engineering. Developed in the early 1990s by Keygene, AFLP uses restriction
enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky ends of
the restriction fragments. A subset of the restriction fragments is then selected to be amplified.
This selection is achieved by using primers complementary to the adaptor sequence the
restriction site sequence and a few nucleotides inside the restriction site fragments (as
described in detail below). The amplified fragments are separated and visualized on denaturing
on agarose gel electrophoresis  either
through autoradiography or fluorescence methodologies or via automated capillary
sequencing instruments.

Although AFLP is commonly referred to as "Amplified fragment length polymorphism", the

resulting data are not scored as length polymorphisms, but instead as presence-absence
polymorphisms.

AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. 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 visualisation of the
band pattern.

Steps:

1.DNA Extraction

DNA extraxted from blood saloiva or other sample and purified.

2.Restriction:
Restriction fragment of the genomic DNA are produced by using two different restriction
enzymes: a frequent cutter (the four base restriction enzyme Mse1) and a rare cutter (the six
base restriction enzyme EcoR1). The frequent cutter serves to generate small fragments, which
amplify well and which have the optimal size range for separation on a sequence gel, whereas
the rare cutter limits the number of fragment to be amplified.

3.ligation of oligonucleotide adaptors:

Double stranded adaptors consist of a core sequence and an enzyme-specific sequence.
Therefore, adaptors are specific for either the EcoR1 site or the Mse1 site. Usually restriction
and ligation take place in a single reaction. Ligation of the adaptor to the restricted DNA alters
the restriction site in order to prevent a second restriction from taking place after ligation has
occurred. The core sequence of the adaptors consist of a known DNA sequence of 20
nucleotides, which will be used later as primer in the PCR.

4.Pre-amplification
This step is a normal PCR where the adapters are used as primers. This first PCR , called
preamplification, allows a first section of fragments by only amplifying the DNA restriction
fragments that have ligated an adapter to both extremities. Additionally to the adapter
sequence, the primer used for pre – selective amplification have a supplementary base. This
extra base enables another first selection by amplifying ¼ of the fragments that have ligated an
adapter to both extremities. These first three steps can be run and visualized on a 1.6% agarose
gel

5.Amplification
The aim of this is to restrict the level of polymorphism and to label the DNA. For
this second amplification, we added 3 ,more nucleotide at the 3 end of primer sequence used
for pre amplification ( adaptor nucleotide + 3 nucleotides) . these two additional nucleotide
makes the amplification more selective and will decrease the number of restriction fragments
amplified .moreover, one of the primers ( usually the EcoR1 primer) is labelled and will allow
the visualization of DNA during the migration

6.Electrophoresis
The PCR products are denatured and run on acrylamide gel ( DNA sequencer ). In our study,
samples were run on an ABI prism 310.
A thin capillary containing a polymer replaces the usual acrylamide gel. The electrophoresis
conditions we used for fragments analysis can resolve DNA fragments differing just by one base
pair . samples are loaded in a track and run one after the other through the capillary.

All fragments are separated with regard to length, smaller fragments running first. Once passing
the laser, a dye attached to primer is excited and emits a fluorescent signal that is then
collected by computer. The results of fluorescence are visualized on the computer as peaks
called Electropherogramms . Each peaks corresponds to a band on a normal acrylamide gel .
amplified fragments range from 30 to 400 base pairs.

Applications:
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.

There are many advantages to AFLP when compared to other marker technologies including
randomly amplified polymorphic DNA (RAPD), restriction fragment length
polymorphism (RFLP), and microsatellites.

 higher reproducibility,
 resolution,
 And sensitivity at the whole genome level compared to other techniques
 , but it also has the capability to amplify between 50 and 100 fragments at one time.
 In addition, no prior sequence information is needed for amplification (Meudt& Clarke
2007).
 As a result, AFLP has become extremely beneficial in the study of taxa including bacteria,
fungi, and plants, where much is still unknown about the genomic makeup of various
organisms.