AFLP AND RFLP STEPS, PRINCIPLE, ADVANTAGE AND DIFFERENCES.

1. Presented By
BIKASH KUMAR SINGH
AFLP & RFLP
1
4/3/2017

2. 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.

3. Procedures in AFLP:
- Digestion
- Adaptor Ligation
- Amplification
- Electrophoresis

4. 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’

5. Restriction site?
Restriction Enzymes: E.g., EcoR1

6. - 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

8. - 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

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

10. RFLP: Restriction Fragment Length Polymorphism

11. RFLP: Restriction Fragment Length Polymorphism

12. RFLP: Restriction Fragment Length Polymorphism

14. Amplified Fragment Length Polymorphism
Double digest (complete)
Ligate adapters – have
complimentary sticky ends
at 3’ end and primer
complimentary sequence
at the 5’ end
Amplify with specific primers
- to amplify only a subset of
fragments formed, the
primers may have 1, 2 or 3
extra nucleotides (shown in
black) beyond the
restriction point (green)
This limits the number of
bands obtained allowing
better resolution and count

15. Arrows indicate two different restriction enzyme cut sites
Complete double digestion
http://www.nature.com/scitable/topicpage/dna-fingerprinting-using-amplified-fragment-
length-polymorphisms-39051
Amplified Fragment Length Polymorphism

16. RFLP based DNA finger printing
Digested DNA Restriction site and VNTR
polymorphism can be
elucidated via southern
hybridization
Hazardous,
Tedious – since at least 10 or more loci
need to be studied
Time taking
Replaced by PCR based finger prints

17. AFLP fingerprints of plant and human DNAs
Each panel shows 3 EcoRI-MseI fingerprints using 3 different
primer combinations. Human DNA fingerprints are displayed in
panel IV, plant fingerprints are displayed in panels I (Arabidopsis),
II (tomato) and III (maize), respectively. Primer combinations are
from left to right:
1. EcoRI+CAA/Msel+CTT
2.EcoRI+CAA/Mse1+CAT
3.EcoRI+CAA/MseI+CTC
4.EcoRl+ACC/Msel+CTT
5.EcoRl+ACC/Mse1+CTC
6.EcoRI+ACC/MseI+CTA
7.EcoRI+ACC/Mse-I+CAT
8.EcoRI+AGG/MseI+CTT
9.EcoRI+AGG/MseI+CAA
10.EcoRI+CAC/Msel+CGA
11.EcoRI+CAC/MseI+CAA
12.E-coRI+CAG/Mse-I+CGA
The molecular weight size range of the fingerprints is from 45 to
500 nucleotides
However, still tedious
involving digestion,
ligation and PCR
Dominant marker

18. AFLP - advantages
Multiple polymorphic restriction sites can be screened in one go
The bands are scored for absence and presence
Unique individual profiles may be developed this way and help in
unambiguous identification of crime suspects or to settle paternity
disputes
Disadvantages –
It is still a three step process involving Digestion, ligation and PCR
Bands from different regions may comigrate to the same distance
because of similar size confounding the analysis

19. •A restriction fragment length
polymorphism (RFLP) is a genetic
variant that can be examined by
cleaving the DNA into fragments
(restriction fragments) with a
restriction enzyme.
WHAT IS RFLP:
19
4/3/2017

20. 20
4/3/2017

21. • Isolating DNA is the first step for many DNA-based
technologies. DNA is found either in nuclear
chromosomes or in organelles (mitochondria and
chloroplasts).
• To extract DNA from its location, several laboratory
procedures are needed to break the cell wall and
nuclear membrane, and so appropriately separate
the DNA from other cell components.
• When doing so, care must be taken to ensure the
process does not damage the DNA molecule and that
it is recovered in the form of a long thread.
Isolating DNA
21
4/3/2017

22. • Extracted DNA is digested with specific, carefully chosen, restriction
enzymes.
• Each restriction enzyme, under appropriate conditions, will
recognize and cut DNA in a predictable way, resulting in a
reproducible set of DNA fragments (‘restriction fragments’) of
different lengths.
• The millions of restriction fragments produced are commonly
separated by electrophoresis on agarose gels. Because the
fragments would be seen as a continuous ‘smear’ if stained with
ethidium bromide, staining alone cannot detect the polymorphisms.
• Hybridisation must therefore be used to detect specific fragments.
Restriction digestion and gel electrophoresis
22
4/3/2017

23. • DNA transfer is called ‘Southern blotting’, after E.M. Southern
(1975), who invented the technique.
• In this method, the gel is first denatured in a basic solution
and placed in a tray. A porous nylon or nitrocellulose
membrane is laid over the gel, and the whole weighted
down.
• All the DNA restriction fragments in the gel transfer as single
strands by capillary action to the membrane. All fragments
retain the same pattern on the membrane as on the gel.
DNA transfer by Southern blotting
23
4/3/2017

24. • The membrane with the target DNA is incubated with the DNA probe.
Incubation conditions are such that if strands on the membrane are
complementary to those of the probe, hybridisation will occur and
labeled duplexes formed.
The DNA probe is a single-stranded molecule, conveniently labeled, using any
standard method (e.g. a radioisotope or digoxygenin), and hybridized with the target
DNA, which is stuck to the membrane.
• Where conditions are highly stringent, hybridisation with distantly
related or non-homologous DNA does not happen.
• Thus, the DNA probe picks up sequences that are complementary and
'ideally‘ homologous to itself among the thousands or millions of
undetected fragments that migrate through the gel.
• Desired fragments may be detected after simultaneous exposure of the
hybridized membrane and a photographic film.
DNA hybridisation: The procedure
24
4/3/2017

25. RFLP technology in pictures
After agarose has been
poured into the gel
mould, combs are
immediately inserted to
form wells and left until
the gel hardens. The
combs are then removed
and the gel placed in an
electrophoresis chamber.
25
4/3/2017

26. Samples of
digested DNA,
with bromophenol
blue dye added,
are loaded into
the wells
with a pipette.
26
4/3/2017

27. After electrophoresis,
the gel is treated with
NaCl to break the
DNA double helix
bonds and make it
single-stranded. This
allows later
hybridisation with a
single-stranded DNA
probe.
27
4/3/2017

28. The blotting tray is
first prepared by
saturating sponges
with NaOH. Safety
glasses and gloves
are required, and a
laboratory coat
recommended.
28
4/3/2017

29. Absorbent paper is placed on top of the
sponges to prevent direct contact with
the gel.
29
4/3/2017

30. Bubbles between the absorbent paper and sponges are removed by rolling
a pipette or a glass rod across the paper. This ensures a complete transfer
of the solution all through
the gel.
30
4/3/2017

31. The treated agarose
gel is placed on top of
the absorbent paper.
31
4/3/2017

32. Membrane is cut
into the appropriate
size.
The membrane is
placed on top of the
gel, then covered
with a piece of
absorbent paper.
32
4/3/2017

33. The entire set-up is topped
with a weight (here, a
bottle of water standing on
a piece of glass) to
promote good transfer.
After some hours the
transfer is complete, the
blotting paper is taken
away, and the membrane
stored until hybridisation
with the probe.
33
4/3/2017

34. The process of hybridisation begins. A DNA is boiled
to denature it to single strands.
34
4/3/2017

35. The labeled probe is
added to the container
with the hybridisation
solution and membrane,
and incubated overnight
in an oven. The
following day, the
membrane is removed
from the hybridisation
set up, and washed with
the appropriate
stringency solution.
35
4/3/2017

36. The membrane is then
blotted dry and put into
a cassette for holding X-
ray film.
The cassette is wrapped,
or sealed with tape, and
stored in a freezer until
the film is
sufficiently exposed,
usually 1 to 4 days.
36
4/3/2017

37. This is an RFLP autoradiogram.
37
4/3/2017

38. RFLPs can be used in many different settings to
accomplish different objectives.
RFLPs can be used in paternity cases or criminal cases to
determine the source of a DNA sample. (i.e. it has forensic
applications).
RFLPs can be used determine the disease status of an
individual. (e.g. it can be used in the detection of mutations
particularly known mutations).
In human population genetics, geographical isolates and
comparison of genetical makeup of related species.
Applications of RFLP:
38
4/3/2017

39. RFLPs can be used in criminal cases
39
4/3/2017

40. • Highly robust methodology with good transferability
between laboratories.
• No sequence information required.
• highly recommended for phylogenetic analysis between
related species.
• Well suited for constructing genetic linkage maps.
• Simplicity—given the availability of suitable probes, the
technique can readily be applied to any plant.
Advantages of RFLPs
40
4/3/2017

41. • Large amounts of DNA required.
• Automation not possible.
• Low levels of polymorphism in some species.
• Time consuming, especially with single-copy probes
• Costly.
• Moderately demanding technically.
Disadvantages of RFLPs
41
4/3/2017

42. 4/3/2017
42
ALFP vs RFLP
AFLP involves selective PCR amplification of
the digested DNA.
RFLP does not involve PCR unless it is PCR-
RFLP.
Sequence Knowledge
Prior sequence knowledge is not required. Prior sequence knowledge is required to
designed RFLP probes.
Reliability
This is more reliable. This is less reliable compared to AFLP.
Efficiency in Detecting Polymorphism
This has a higher efficiency in detecting
polymorphism than RFLP.
This is less efficient compared to AFLP.
Cost
This is a little expensive compared to RFLP. This is less expensive compared to AFLP.
Applications
AFLPs have been applied to genome mapping,
DNA fingerprinting, genetic diversity studies,
paternity testing and forensics
RFLP analysis is an important tool in genome
mapping, localization of genes for genetic
disorders, determination of risk for disease,
and paternity testing.
What is the difference between AFLP and RFLP?

43. 4/3/2017
43
• `1. Garcia, Antonio A. F., Luciana L. Benchimol, Antônia M. M. Barbosa,
Isaias O. Geraldi, Souza Jr. Cláudio L., and Anete P. De Souza.
“Comparison of RAPD, RFLP, AFLP and SSR markers for diversity studies
in tropical maize inbred lines.” Genetics and Molecular Biology.
Sociedade Brasileira de Genética, 2004. Web. 19 Mar. 2017
• 2. “Restriction Fragment Length Polymorphism (RFLP).” National Center
for Biotechnology Information. U.S. National Library of Medicine, n.d.
Web. 19 Mar. 2017
• 3. Masiga D. K. and Turner C. M. (2004). “Amplified (restriction)
fragment length polymorphism (AFLP) analysis”. Methods Mol Biol:
270: 173-86. NCBI. Web. 19 Mar. 2017
Reference: