KT đa hình các đoạn phân cắt...AFLP

AFLP
Amplified Fragment Length Polymorphism
AFLP
- a method based on PCR developed in 1995 by Vos et al.
Involves the use of RFLP and PCR techniques
Compared with the widely used RFLP, AFLP is faster, less labour intensive and provide more information.
An additional advantage over RAPD is their reproducibility.
AFLP
The AFLP technique is based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
AFLP
Procedures in AFLP:
- Digestion
- Adaptor Ligation
- Amplification
- Electrophoresis
Two different restriction endonucleases are used in digestion. One is 4-base cutter (MseI) and the other one is 6-base cutter (EcoRI).
Digestion
MseI 5’TTAA3’
EcoRI 5’GAATTC3’
Two different adaptors (short double stranded DNA sequences with sticky end) are ligated to the digested fragments.
One adaptor will complement to the Msel cut end, the other will complement to the EcoRI cut end.

Adaptor Ligation
DNA fragments with MseI-EcoRI ends with be selected as DNA template for amplication.
two PCR primers complementary to the two adaptors are used in amplification.
the PCR primers are labelled with radioactive or fluorescence dye for detection of DNA bands on gels.
Amplification
polyacrylamide gel is used for separating DNA bands.
Normally, 30-100 DNA bands can be detected by AFLP on polycrylamide gel.
Electrophoresis
The number of DNA bands detected by AFLP is high. It can be reduced by adding selective bases (1-3 nucleotides) at the 3’-end of the PCR primers.
one additional selective base on the primer can reduced the number of DNA bands 16 folds.
three additional selective bases on the primer can reduce the number of DNA bands 4,000 folds.
Selective Bases
Polymorphism among 32 wheat samples revealed by AFLP
dominant marker.
DNA variation is detected by presence/absence of DNA bands due to:
a) presence/absence of restriction sites
b) additional bases (insertion) between two restriction sites are too large
Characteristics of AFLP
higher reproducibility compared to RAPD.
highly polymorphic.
Advantages