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Rapd and aflp - Rapd and Rflp
Biotechnology (BCH 501)
University of Kerala
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RAPD
KAPD
stands
for
Random
Amplification
of
Polymorphic DNA.
It is
a
type
of
####### PCR,
but the
seg
O DNA that are amplified are random. Unlike traditional PCR analysis, RAPD oes
dny
####### specific
knowledge
of
the
DNA
sequence
of the target organism.
In
KAFD
dr
####### DErary,
short
####### primers
(8-
nucleotides) and a
####### large
template
of
genomic
DNA
a
O
Knowledge of
the
DNA
sequence
for the
targeted
gene
is
required,
as the
prie
Od
####### somewhere
in the
sequence, but it is not
certain
exactly
where. This makes
tne
e
Popuidr ror
comparing the
DNA of
####### biological
systems
that have not
had the attention
or ne
Sentiic
community,
or in a
####### system
in which
####### relatively
few DNA
####### sequences
are
####### compared.
1n
recent
####### years, RAPD
has
been used to
####### characterize and
trace,
the
phylogeny
of diverse plant
not
will
hod
and animal species.
RAPD
Involves
amplificationof
DNA
####### fragmentsfrom
any
species by
use of a single
arbitrdry
####### Oligonucleotide
####### primer
without
prior sequence information.
As the
approach
####### requires
no
prO
Knowiedge of the genome that is being analyzed, it can be employed across species using
universal
primers. The
####### major limitation of the
RAPD method is the
reproducibility
and dominant
inheritance. Several factors influence the reproducibility of RAPD reactions such as quality
and quantity of template DNA, PCR buffer, concentration of magnesium chloride, primer to
emplate ratio and annealing temperature. RAPD markers are dominant markers, and hence
do not
distinguishdominant
homozygotes
from
heterozygotes.
2
Sample 1 dsDNA
5
Unlike traditional
PCR PCR analysis, RAPD
####### (pronounced'rapid')
does not require any
specific knowledge of
the DNA sequence of
Product A
Product B
the target organism:
the identical10-mer
primers will or will not dsDNA
Sample 2
amplify a segment
of DNA, depending
PCR
on positions that are
complementary to the
primers' sequence. For
Product B
Example, no fragment
5
produced
if
####### primers
annealed too far apart
or
3
-ends
of the
primers
àre not facing each
other, Therefore, if a
Figure
Schematic
drawing
of
reaction
conditions
for RAPD. The primers
must anneal in a particular
orientation (such
that they
point
towards
each other)
and
within a reasonable
distance of one another. The
arrows represent
multiple
copies
of a single
primer
and
the
direction of the arrow indicates the direction
in
which
DNA synthesis
will
ocCur. The
numbers represent
primer annealing
sites on
the DNA template.
For sample
1,
primers
anneal
to
sites 1, 2,
and 3 on
the top
strand of the DNA
template
and to sites 4,
5,
and 6
on
the
bottom
strand
of the
DNA template.
In this example, only
2 RAPD
products
are formed.
For sample
1:
(i)
product
A is produced
by
PCR amplification
or the DNA sequence which lies in
between
the primers
bound
at positions
2 and 5;
and (i)
product
5 is produced by
PCR amplification of the
DNA
equence
which
lies in
between
the primers
bound
at positions
3 and 6. No PCR product is
produced by
the
primers
bound
at positions
1 and
4 because
these primers
are too tar
apart
to allow completion
of the
PCR
reaction.
No
PCR products
are
also produced
by
the primers
bound at positions 4 and 2 or
positions
5
and
3
because
these primer
pairs
are not
oriented
towaras each other. For
sample 2,
the primer
failed to
anneal
at position
2
and
PCR product
was
obtained ony
ror primers bound at position
3 and
mutation has occurred in
the template DNA at the
Ste
that was
previously
omplementary to the
####### primer, a PCR product
will
not be
produced
fesultingin a different
pattern of amplified DNA
####### Segments on the
ge
Molecular Biology Techniques 161
To
overcome
the
limitation
of
reproducibility
associated
with RAPD,
AFLP (Amplified
Fragment
Length
Polymorphism)
technology
was
developed.
Like RAPD,
AFLP
does
not require any
DNA
sequence
information
from
the
organism
under study
and
also a
dominant
marker. However, it
is highly
reliable
and
reproducible.
It
combines
the power
of
RFLP
with the flexibility
of
PCR
based
technology
####### by
ligating
primer
recognition
sequences
(adaptors)
to the
restricted DNA
AFLP
limited set of primers. The
and
selective
PCR
amplification
of
restriction
fragments
using
first step
in
AFLP analysis
involves
restriction digestion
of genomic
DNA
with a combination
of
rare
cutter
####### (EcoRI
or PstI)
and frequent
cutter (Msel
or TaqI)
restriction enzymes.
Double-
stranded
oligonucleotide
adaptors
are,
then,
designed
in
such a way
that the
initial restriction
site is
not
restored
after ligation.
Such adaptors
are ligated
to
both ends
of the fragments
to
provide
known sequences
for
PCR
amplification.
PCR
amplification
will only
occur where
the
primers
are able
to
anneal
to fragments
which
have
the adaptor
####### sequence plus
the comple-
mentary
base pairs
to
the
additional
nucleotides
called
selective
nucleotides.
An aliquot
is
then subjected
to two subsequent
PCR amplifications
under
####### highly
stringent
conditions with
primers
complementary
to the adaptors,
and possessing
3'
selective
nucleotides of 1-
bases.
Restriction site for EcoRI
Restriction site for Msel
####### =TTAA
AATT
=GAATTC
-CTTAAG
Digestion
with
restriction enzymes
EcoRI
and Msel
AAT
####### AATTC =
=CTTAA +
AATTG
Msel adaptor
C
EcORI adaptor
= GTAA-
=CATT=
GAATTG
CTTAAC
CATTGTA
G TAA
CATT=
MseI primer 1-
-GAATTG
CTTAAC
CGAGA ATTG
EcORI primer 1
Figure 12 AFLP procedure. The procedure of this technique is divided into two steps:
- Digestion of cellular DNA with two restriction enzymes and ligation of restriction half-site specific
adaptors. The adaptor is designed in such a way that ligation of a fragment to an adaptor does not
reconstitute the reaction site.
- Selective amplification of some of these fragments with two PCR primers that have corresponding
adaptor and restriction site-specific sequences
To achieve selective amplification of a subset of these fragments, primers are extended into the un-
known part of the fragments [underlined base], usually one to three arbitrarily chosen bases beyond
the restriction site. The first is performed with a single-bp extension, followed by a more selective
primer with up to a 3-bp extension. Because of the high selectivity, primers differing by only a single
base in the AFLP extension amplify a different subset of fragments.
Rapd and aflp - Rapd and Rflp
Course: Biotechnology (BCH 501)
University: University of Kerala
