Amplified Fragment Length Polymorphism (AMP-FLP)

Amplified Fragment Length Polymorphism (AMP-FLP)

A range of DNA extraction methods has been used for forensic DNA analysis. For example, digestion of body fluid stains using SDS and proteinase K, followed by purification of DNA by extraction with phenol/chloroform and ethanol precipitation, is very successful and is routinely used for forensic samples analyzed by RFLP typing. However, this method had limitations when applied to a PCR-based DNA typingmethod for forensic analysis viz. HLA DQα typing. For bloodstains, it was observed that, although adequate DNA was obtained for analysis, it could not always amplify using PCR. This failure in amplification process is found to be caused by the existence of hematin in bloodstains, since hematin is an inhibitor of PCR.

Another PCR-based DNA typing method, used for the analysis of amplified fragment length polymorphisms (AMP-FLPs) could be implemented in forensic laboratory but it was advantageous to assess a number of DNA extraction methods to decide the most suitable one for AMP-FLP analysis. The AFLP method was developed in 1995 by Vos et al. and has been used for numerous years in research laboratories and for patent applications.Factors considered, when various methods were compared include the yield of DNA, the suitability of DNA for amplification, existence of fragments of DNA on a silver stained gel and the differential amplification of alleles having different sizes in a sample. A variety of extraction methods was experimented for all these factors, including Chelex100 extraction, organic extraction followed by either ethanol precipitation or Centric on 100® dialysis and concentration and several commercially available DNA extraction kits.

The features of optimized AFLP analysis project the assay valuable for a number of clinical applications. The human identity testing has evolved from agarose gel-based separation of DNA restriction fragments to capillary electrophoresis platforms usage and this move has greatly improved the resolution of the separation technique.

AFLP is an excellent technology to be used in the detection, separation, and ascriptionof a microbial strain in the case of a bio crime. Several forensic cases include plant evidence that may be valuable to link a victim, a suspect, a vehicle, a weapon and crime scenes. With the introduction of novel DNA technologies, plant DNA material can be chemically extracted and typed using a multilocus detection methodcalled AFLP. It is a PCR-based method to produce DNA fingerprints and speedily screen genetic diversity. AFLP uses a pair of restriction enzymes to cut up the genomic DNA unlike markers such as microsatellites, where designed primers target the markers in the genome. Then, a pair of synthetic DNA fragments, adaptors, is attached to the complementary sticky ends of genomic DNA fragments during the ligation phase. It is followed by pre-selective and selective PCR stages, which use primers that match the known adaptor sequence along with additional selective nucleotides, which increase the specificity of amplification, thus reducing the total number of final fragments or loci. In the selective PCR stage, one of the primers having a fluorescent label attached allows the DNA fingerprints to be visualized by electrophoresis using a sequencer. Fig.3 shows the use of the adaptors/primers Eco and Pst and loci derived from 6 primer combinations i.e. selective primers with different selective nucleotides.