AFLP is a PCR-based DNA fingerprinting technique that uses restriction enzymes to digest genomic DNA and ligate adaptors to the restriction fragments. Selected fragments are amplified using primers complementary to the adaptors. This yields highly specific and reproducible genotypic data that can be used for applications like monitoring inheritance of traits, disease diagnosis, forensic analysis, and species identification. AFLP provides polymorphic profiles even with small amounts of DNA without requiring prior genome knowledge.

1. A F L P
Amplified Fragment Length
Polymorphism
Jewell Ann P. Manabat
MS Biology Education
Advanced Biotechnology

2. AFLP
 or AFLP-PCR
 a PCR-based tool
 developed by Keygene
in the early 1990s

3. AFLP
 uses restriction enzymes to
digest genomic DNA
 ligation of adaptors to the
sticky ends of restriction
fragments
 amplification of selected
subset of the restriction
fragments (60-500 bp)
 higher repeatability
compared to RAPD and ISSR

4. AFLP
 even small amounts of
genomic DNA can be used
to produce DNA fingerprints
that are highly specific to
particular species
 does not require any prior
of the genome sequence

5. AFLP
 uses many of the same
steps as the other markers
(RFLP, SSR, RAPD)
 includes additional steps
that permit high resolution
interrogation of the entire
genome
 yields highly specific,
reproducible genotypic data

6. Steps:
1. Digestion
2. Adaptor ligation
3. Amplification
4. Electrophoresis

8. 1. Digestion
 two restriction enzymes:
- MseI
* 4-base cutter
- EcoRI
* 6-base cutter
MseI 5’TTAA3’
EcoRI 5’GAATTC3’

9. Restriction Enzymes
 Found in bacteria
 Cut DNA within the molecule (endonuclease)
 Cut at sequences that are specific for each enzyme
(restriction sites)
 Leave either blunt or sticky ends, depending upon the
specific enzyme

10. 2. Adaptor Ligation
 2 different adaptors
- short double stranded
DNA sequences
- with sticky ends
- complements the REs

11. 3. Amplification
 DNA fragments with MseI-
EcoRI ends will be selected
 two PCR primers
complementary to the
adaptors
 primers are labelled with
radioactive or fluorescent
dyes

12. 4. Electrophoresis
 polyacrylamide gel
 detects 30-100 DNA bands

14. Selective bases
 added at the 3’-end of the
primers
 1-3 nucleotides
 can reduce the number of
DNA bands
1 nucleotide – up to 16 folds
3 nucleotides – up to 4000 folds

15. Genotyping
If there are 2 new priming
sites within 400-1600bp =
amplification
 result = presence or
absence of amplification
 mostly due to SNP
 also deletions or insertions

16. Advantages
 replaces RFLP in
fingerprinting technique
 highly polymorphic
 high reproducibility
 identify through absence or
presence of fragment
 characters can be increased
by changing the restriction
enzyme and nucleotide at
selective primers

17. Disadvantages
 Dominant – lose the
codominant character
 Homology – ability to
differentiate different
fragment with similar size
 Mutation rate – high
homoplasy
- High levels of variation
 Scoring - bias

18. Applications
 monitoring inheritance of
agronomic traits
 diagnostic in genetically
inherited disease
 pedigree analysis
 forensic typing (parentage
analysis)
 identifying hybrids
 species level relationship