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Advanced Biotechnology

AFLP
Amplified Fragment Length
Polymorphism

Jewell Ann P. Manabat


MS Biology Education
AFLP
 or AFLP-PCR
 a PCR-based tool
 developed by Keygene
in the early 1990s
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
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
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
Steps:
1. Digestion
2. Adaptor ligation
3. Amplification
4. Electrophoresis
1. Digestion
 two restriction enzymes:
- MseI
* 4-base cutter
- EcoRI
* 6-base cutter
MseI 5’TTAA3’
EcoRI 5’GAATTC3’
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
2. Adaptor Ligation
 2 different adaptors
- short double stranded
DNA sequences
- with sticky ends
- complements the REs
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
4. Electrophoresis
 polyacrylamide gel
 detects 30-100 DNA bands
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
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
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
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
Applications
 monitoring inheritance of
agronomic traits
 diagnostic in genetically
inherited disease
 pedigree analysis
 forensic typing (parentage
analysis)
 identifying hybrids
 species level relationship

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