This post we shall discuss one of the widely used technique for DNA fingerprinting.
AFLP or Amplified fragment length polymorphism is a highly sensitive method DNA fingerprinting technique, that is, it is used for detecting polymorphisms in the given sample of DNA.
The original concept was brainchild of Vos and Zabeau in 1993 and combines the principles of two different techniques namely; Restriction Length Fragment Polymorphism (RFLP) and Polymerase Chain Reaction (PCR).
In this, the DNA sample is first cut using restriction enzymes, followed by ligation of complementary double stranded adaptors to the ends of the restriction fragments. A subset of the ligated restriction fragments is then amplified using two primers complementary to the adaptor and restriction site fragments. The fragments are run and visualized on denaturing polyacrylamide gels either through autoradiography or fluorescence methodologies (labelled primers) and analyzed
This entire procedure can be divided into four main steps:
(a) Restriction of DNA sample:
The DNA used is large DNA like genomic DNA (gDNA), hence more than one type of restriction enzymes are used. Each may differ in their frequency of cutting the DNA, that is the distribution of their restriction sites in the DNA may vary.
Example: let us consider in our case EcoR1 and Mse1. Both these restriction enzymes produce sticky ends (overhanging ends). In case of EcoR1 and Mse1, EcoR1 (6 base pair long restriction site) is a rare cutter and Mse1 is a frequent cutter (4 bps long restriction site).
(b) Ligation of adaptors to ends of the digested fragments:
The double-stranded adaptors are then attached to the either ends of the cleaved DNA fragments (most of the times, step a an b are performed together). Adaptors are chemically synthesized (i.e. of known sequence) linker oligonucleotides with sequence homologous to the restriction site. Their sequences are known and they flank the DNA fragments, hence used to design primers, in the next step of PCR amplification.
(c) PCR amplification:
The restricted fragments ligated with adapters are then amplified. The primers are designed complementary to (and they anneal to) the adaptors and restriction site.
As the gDNA is very large and May generate a very large number of restriction fragments, selective amplification of only a subset of fragments is carried out. The selectivity is achieved by designing PCR primers that anneal specifically to the adaptor, recognition site and has one to three (or even five) arbitrary chosen nucleotides at the 3′ end. Only those restriction fragments will be amplified that have adapter region, the recognition site and complementary nucleotides to those ‘arbitrary nucleotides sequence’.
There are two levels of selectivity carried out in two different steps:-
i. Preselective Amplification:-
In the first amplification step, that is the Preselective Amplification, a subset of the restricted fragments are only amplified. The primer is used is complementary to the sequence of adapter, the restriction site and plus one nucleotide. Hence the fragments having the particular complementary base pair will only be amplified. Doing this reduces the bulk of DNA and ‘selects’ only a subset of fragments. The nucleotide chosen has to be decided on the basis of the final electrophoretic results.
Fig 2: Preselective Amplification in AFLP using +1 nucleotide (here C)
In the fig 2: the primer is complementary to the adaptor, the remaining part of the EcoRI recognition site and an extended ‘C’. So all the fragments having the adapter part, recognition site of EcoRI and a ‘G’ will only be amplified. Same will be the case for the other restriction sites.
ii. Selective amplification:
The subset of the restriction fragments amplified from first step are further selectively amplified by adding more two nucleotides to the primer (that is adaptor + restriction site + 3 nucleotides primer). Hence the number of fragments amplified are further reduced.
In the example given (fig 3) for the selective amplification +3 nucleotide primers (CTA) has been used, so all the fragments having GAT next to the restriction site will only be amplified. This reduces the number of fragments amplified making results legible, and not too crowded.
The sequence of the primers have to be carefully standardised after analyzing the results from different primer designs (with different nucleotide combination). The primers can be labelled, either with fluorescent or radioactive molecule.
(d) Electrophoretic Separation:
The selected subset of the fragments amplified are then separated by electrophoresis to give a DNA profile. Any SNP or mutation which affects the presence or absence of restriction site causes polymorphism in the length of the band, which can be detected in the gel. Results can be analysed using various softwares.
Here’s an example of such an AFLP result.
- High reproducibility
- High resolution
- Genomic DNA can be used
- Can amplify between 50 and 100 fragments at one time.
- No prior sequence information is needed for amplification.
The AFLP technology can detect various polymorphisms in different genomic regions simultaneously.
AFLP has been 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
AFLP provides great information in population genetics to determine slight differences within populations and in linkage studies to generate maps for QTL analysis.
AFLP has been proved to be advantageous than other marker based techniques like randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and microsatellites.
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