6-5.3(a).1.3 Amplified Fragment Length Polymorphism (AFLP)

►  AFLP is also known as selective restriction fragment amplification (SRFA) as it involves selective PCR amplification of restriction fragments from a total double digest of genomic DNA under stringent condition.

►  AFLP is more efficient than RFLP & RAPDin detection of polymorphism and has high reproducing ability.

►  Most AFLPs are well known as dominant markers.

►  The AFLP technique involves both the previous mapping basics such as restriction digestion of RFLP and PCR amplification of RAPD.

►  The additional step in AFLP is ligating the fragments with adaptors of known sequence against which the primer has to be designed followed by PCR.

►  One of thelimitations of AFLP is that it is too complicated to be suitable for genotyping or MAS (Marker assisted selection).

 

AFLP comprises of three main steps (Figure 6-5.3(a).1.3) (Vos et al., 1995).

1.  Cutting of genomic DNA with two restriction endonuclease enzymes simultaneously.

2.  Ligation of adapters to the genomic DNA fragments to generate target sites for primer annealing and selective amplification of a subset of genomic restriction fragments by polymerase chain reaction (PCR). PCR amplification involves two consecutive reactions. In first PCR reaction (pre-amplification) DNA fragments are amplified with two AFLP primers, each having one selective nucleotide. In the second PCR reaction (selective amplification) the products are diluted and used as templates for the amplification using two AFLP primers, each having three selective nucleotides.

3.  Separation and detection of the amplified fragments DNA using denaturing polyacrylamide gel electrophoresisand silver staining respectively.