AFLP (Amplified Fragment Length Polymorphism ) description
- 1. AFLP AFLP Amplified Fragment Length Polymorphism Amplified Fragment Length Polymorphism
- 2. AFLP AFLP - a method based on PCR developed in 1995 by Vos et al. - Involves the use of RFLP and PCR techniques - Compared with the widely used RFLP, AFLP is faster, less labour intensive and provide more information. - An additional advantage over RAPD is their reproducibility.
- 3. AFLP AFLP The AFLP technique is based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
- 4. AFLP AFLP Procedures in AFLP: - Digestion - Adaptor Ligation - Amplification - Electrophoresis
- 6. Two different restriction endonucleases are used in digestion. One is 4-base cutter (MseI) and the other one is 6-base cutter (EcoRI). Digestion Digestion MseI 5’TTAA3’ EcoRI 5’GAATTC3’
- 8. - Two different adaptors (short double stranded DNA sequences with sticky end) are ligated to the digested fragments. - One adaptor will complement to the Msel cut end, the other will complement to the EcoRI cut end. Adaptor Ligation Adaptor Ligation
- 10. - DNA fragments with MseI-EcoRI ends with be selected as DNA template for amplication. - two PCR primers complementary to the two adaptors are used in amplification. - the PCR primers are labelled with radioactive or fluorescence dye for detection of DNA bands on gels. Amplification Amplification
- 11. - polyacrylamide gel is used for separating DNA bands. - Normally, 30-100 DNA bands can be detected by AFLP on polycrylamide gel. Electrophoresis Electrophoresis
- 13. - The number of DNA bands detected by AFLP is high. It can be reduced by adding selective bases (1-3 nucleotides) at the 3’- end of the PCR primers. - one additional selective base on the primer can reduced the number of DNA bands 16 folds. - three additional selective bases on the primer can reduce the number of DNA bands 4,000 folds. Selective Bases Selective Bases
- 14. Polymorphism among 32 wheat samples revealed by AFLP
- 15. - dominant marker. - DNA variation is detected by presence/absence of DNA bands due to: a) presence/absence of restriction sites b) additional bases (insertion) between two restriction sites are too large Characteristics of AFLP Characteristics of AFLP
- 16. - higher reproducibility compared to RAPD. - highly polymorphic. Advantages Advantages