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AFLP: A Guide to DNA Fingerprinting

This document discusses AFLP (Amplified Fragment Length Polymorphism), a PCR-based technique for fingerprinting genomes using restriction enzymes. It involves digesting genomic DNA with restriction enzymes, ligating adaptors to the restriction fragments, selectively amplifying subsets of fragments between 60-500 base pairs via PCR with primers complementary to the adaptors, and analyzing the amplified fragments via electrophoresis to generate highly reproducible DNA fingerprints specific to species. AFLP permits interrogation of the entire genome and yields highly specific genotypic data useful for applications like monitoring trait inheritance, disease diagnosis, forensic analysis, and studying species relationships.

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8 views19 pages

AFLP: A Guide to DNA Fingerprinting

This document discusses AFLP (Amplified Fragment Length Polymorphism), a PCR-based technique for fingerprinting genomes using restriction enzymes. It involves digesting genomic DNA with restriction enzymes, ligating adaptors to the restriction fragments, selectively amplifying subsets of fragments between 60-500 base pairs via PCR with primers complementary to the adaptors, and analyzing the amplified fragments via electrophoresis to generate highly reproducible DNA fingerprints specific to species. AFLP permits interrogation of the entire genome and yields highly specific genotypic data useful for applications like monitoring trait inheritance, disease diagnosis, forensic analysis, and studying species relationships.

Uploaded by

Teflon Slim
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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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