aflp-200507044155.ppt for student education

Information for student about Biochemistry

1. AFLP MARKER
By
Ishrat Fatima
Department of Botany
University of Agriculture, FaisalabadUniversity of Agriculture, Faisalabad

2. Contents
Introduction
Steps in AFLP
Advantage of AFLP
Disadvantage of AFLP
Application
Conclusion

3. Amplified fragment length polymorphism
AFLP method based on PCR developed in 1995 by
Vos et al.
- Involves the use of RFLP and RAPD techniques and
universally used.
- Compared with the widely used RFLP, AFLP is
faster, less labour intensive and provide more
information.
- An additional advantage over RAPD is their
reproducibility.

4. Amplified fragment length
polymorphism
This is highly sensitive method for detecting
polymorphism throughout the gnome and is becoming
increasingly popular.
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.

5. STEPS IN AFLP
Digestion
Adaptor Ligation
Amplification
Selective amplification
Electrophoresis
Data collection

6. DIGESTION
Two different restriction endonucleases are used in
digestion. One is 4-base cutter (MseI) and the other one
is 6-base cutter (EcoRI).
GAATTC
CTTAAG
AATT
EcoR I Mse I
AATTC
G
T
AAT
TTAA

7. ADAPTOR LIGATION
 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.
AATTC
G
T
AAT
EcoR I
adapter
Mse I
Adapter
AATTC
TTAAG
TTA
AAT

8. AMPLIFICATION
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.
AATTC
TTAAG
TTA
AAT
AATTC
TTAAG
TTA
AAT

9. SELECTIVE
AMPLIFICATION
Selective Bases -
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.

10. SELECTIVE
AMPLIFICATION
AATTCN
TTAAGN
NTTA
NAAT
AATTCA
TTAAGT
GTTA
CAAT
AATTCAAC
TTAAGTTG
TTGTTA
AACAAT
A
C
AAC
AAC

11. ELECTROPHORESIS
 polyacrylamide gel is used for separating DNA
bands.
 Normally, 30-100 DNA bands can be detected by
AFLP on polycrylamide gel.

12. GAATTC
CTTAAG
TTAA
AATT
EcoR I Mse I
AATTC
G
T
AAT
EcoR I
adapter
Mse I
Adapter
AATTCN
TTAAGN
NTTA
NAAT
AATTCA
TTAAGT
GTTA
CAAT
AATTCAAC
TTAAGTTG
TTGTTA
AACAAT
A
C
AAC
AAC
polyacrylamide gel electrophoresis

13. DATA COLLECTION
Instrument for electrophoretic separation and fluorescence detection
During the electrophoresis, the instrument monitors the passage the
fluorescent, 5' dye-labeled fragments through the polymer by
detecting fluctuations in emitted light when the fragments migrate
past a fixed laser beam.
When finished, the instrument assembles the spectral data for each
sample and stores it a sample file, or saves it to the application
database.

14. ADVANTAGE
This technique is extremely sensitive.
It has high reproducibility proving extremely proficient
in revealing diversity.
It discriminates heterozygote's from when a gel scanner
is used.
It permits detection of restriction fragments in any
background or complexity including pooled DNA
samples and colned DNA segments.
It not a simple fingerprinting technique but it can be used
for mapping also.

15. DISADVANTAGE
It is extremely expensive and requires more DNA
than is needed is RAPD.
 It is technically more demanding than RAPSs as
it requires experience of sequencing gels.
 AFLPs are expensive to generate as silver
staining, fluorescent dye or radioactivity detects
the bands.

16. APPLICATION
To detect various polymorphisms in different genomic regions.
For the identification of genetic variation in strains or closely
related species of plants, fungi, animals, and bacteria.
 The AFLP technology has been used in criminal and paternity
tests.
 In linkage studies to generate maps for qualitative trait
locus
 (QTL) analysis.