AMPLIFIED FRAGMENT LENGTh POLYMorphism
(AmpFLP)
A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps:
(i) Restriction of the DNA and ligation of oligonucleotide adapters,
(ii) Selective amplification of sets of restriction fragments, and
(iii)Gel analysis of the amplified fragments.
PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence.
The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing Polyacrylamide Gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity. [28] & [19]
Process for AFLP is explained below-
PROCESS-
- AFLP analysis starts with a sample of genomic DNA isolated and purified from a single individual. After the genomicDNA is obtained, it is digested with a pair of Restriction Enzymes, often MseI and EcoRI .
- MseI recognizes 5′-TTAA-3′ and cleaves after the first 5′-T, whereas EcoRI recognizes 5′-GAATTC-3′ and cleaves after the 5′-G. The genomic DNA is typically incubated with these enzymes for a period long enough to permit complete digestion of the DNAinto restriction fragments.
- In order to make use of the fragments of genomic DNA generated by MseI and EcoRI digestion, an MseI adaptor and an EcoRI adaptor, each of which is a short, double-strandedDNA molecule with a 5′ overhang that is complementary to the 5′ overhang was generated by MseI (5′-TA-3′) and EcoRI (5′-AATT-3′), respectively.
- Rather than containing a 3′-A after the 5′-TA-3′ overhang, the MseI adaptor contains a 3′-C, which destroys the MseI recognition site once ligated.
- Similarly, rather than containing a 3′-C after the 5′-AATT-3′ overhang, the EcoRI adaptor contains a 3′-G, which destroys the EcoRI recognition site once ligated.
- The MseI and EcoRI adaptors each contain their own specific DNA sequence, typically between 19 and 22 base pairs in length, that is located upstream of their respective 5′ overhangs.
- The aforementioned ligation reaction involves incubation of the MseI/EcoRI digested genomic DNA together with the two adaptors and with DNA ligase, which is an enzyme that covalently links the adaptors with their respective complementary 5′ ends.
- MseI and EcoRI restriction enzymes can also be added to this ligation reaction, as they will prevent re-ligation between EcoRI-EcoRI and MseI-MseI genomic fragments and will not recognize the ligated adaptor-genomic DNA fragments. [2J] & [3J]
- After ligating the genomic DNA fragments with their corresponding adaptors, researchers are left with a series of DNAfragments that consist of the specific MseI adaptor-associated sequence, the destroyed MseI site, a genomic DNAfragment, the destroyed EcoRI site, and the specific EcoRI adaptor-associated sequence. Therefore, each unique genomic DNA fragment is sandwiched between the MseI adaptor and the EcoRI adaptor and their associated uniqueDNA sequences.
BUT.... HOW TO SELECT THE TARGET GENE????
- Although the MseI and EcoRI adaptor-ligated ends of each DNA fragment will be identical, the sequences of the genomic DNA fragments, which begin just after the original MseI and EcoRI sites, will differ.
- If PCR was carried out, primers corresponding to the MseI and EcoRI adaptor sequences would amplify every single genomic DNA fragment.
- Thus, in order to selectively amplify a smaller number of genomic DNA fragments, researchers use primer sets that are complementaryto the MseI or EcoRI adaptor sequences starting at their 5′ ends but that add up to three unique nucleotides following the end of the original MseI or EcoRI recognition site.
- The addition of only a small number of base pairs, called selective nucleotides, at the end of each primer permits the amplification of a subset of genomic DNA fragments.
- The more complex the genome being investigated, the more selective nucleotides researchers add to the primers. Some simpler genomes might not require any selective nucleotides at all as shown in the figure above.
- Hence, As the number of nucleotides added to the 3′ end of each primer is increased, the selectivity of the primer also increases. [2J] & [3J]
- The PCR reactions carried out during the third step of the AFLP-PCR process will simultaneously amplify a wide range ofDNA fragment sizes, based on the length of the original MseI-EcoRI genomic DNA fragment ligated to the MseI and EcoRI adaptors.
- In order to determine the AFLP-PCR-derived DNA fingerprint, researchers must be able to identify and characterize the set of DNA fragments produced in a given PCR reaction.
- Thus, in most cases, one of the two PCR primers is radioactively or fluorescently labeled (typically the EcoRI primer), which leads to the production of a labeled PCR product that can be easily detected. [2J] & [3J]