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

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A f l p

  • 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