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.
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
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
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