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RAPD (Random Amplified

Polymorphic DNA)
RAPD (Random Amplified Polymorphic DNA)

 Teknik RAPD mendeteksi polimorfisme urutan nukleotida


dari fragmen DNA hasil amplifikasi PCR dengan
menggunakan satu primer tunggal.

 Dalam reaksi ini satu jenis primer akan berikatan dengan


pasangan sekuen komplemennya di dua tempat yang
berbeda pada dua utas yang berlawanan di DNA genom
yang sebelumnya telah terdenaturasi. Jika penempelan
primer yang satu dengan yang lain berada pada jarak
yang dapat diamplifikasi maka penggandaan fragmen
DNA secara in-vitro dapat diperoleh.
 Amplifikasi sekuen DNA genom pada mesin PCR
melibatkan pengaturan temperatur yang terjadi
secara berulang.
 Reaksi terdiri dari denaturasi DNA menjadi utas
tunggal (temperatur 94ºC), penempelan primer
(annealing) pada sekuen DNA genom (temperatur
25º-65ºC), dan pemanjangan primer (elongation)
pada temperatur 72ºC.
 Pengulangan siklus 25-50 kali akan meningkatkan
jumlah fragmen DNA yang diamplifikasi secara
eksponensial
The polymerase
chain reaction
(PCR)
A discrete PCR product is produced when, at an appropriate annealing
temperature, the single primer binds to sites on opposite strands of the
genomic DNA that are within an amplifiable distance (Figure 5),
generally less than 3,000 base pairs.
RAPD technology
A B C
A

+ +
+

Taq polymerase Arbitrary primers Nucleotides

+
Genomic DNA

PCR

(under relaxed conditions)

Buffer
Arbitrary Primer
 The RAPD protocol usually uses a 10 bp arbitrary primer at
constant low annealing temperature (generally 34 – 37 °C).
 RAPD primers can be purchased as sets or individually from
different sources, such as the University of British Colombia
(http://www.michaelsmith.ubc.ca/services/
NAPS/Primer_Sets) and the Operon Biotechnologies
(http://www.operon.com).
 Two basic criteria indicated by Williams et al. (1990) must be
met: a minimum of 40% GC content (50 - 80%) GC content
is generally used) and the absence of palindromic sequence
 Because G-C bond consists of three hydrogen bridges and
the A-T bond of only two, a primer-DNA hybrid with less than
50% GC will probably not withstand the 72 oC temperature at
which DNA elongation takes place by DNA polymerase.
 The resulting PCR products are generally resolved on
1.5-2.0% agarose gels and stained with ethidium
bromide (EtBr); polyacrylamide gels in combination
with either AgNO3 staining (e.g., Huff et al., 1993; Vejl,
1997;Hollingsworth et al., 1998)
PCR

360 bp

Electrophoresis
A B C

260 bp
520bp

520 bp 360 bp

A B C 260 bp
PCR product occurs when:
 The primers anneal in a particular orientation
(such that they point towards each other)
 The primers anneal within a reasonable
distance of one another (150 -3000 bp)
The number of amplification products is related to the
number and orientation of the genome sequences which are
complementary to the primer

1 2 3

4 5 6

DNA template PCR reaction

Product 1 Product 2
The nature of RAPD
polymorphism
a) nucleotide substitution within target sites may affect
the annealing process - either no fragment is detected

1 3

4 5 6

DNA template PCR reaction

No product
Product 2
or detected fragment is of increased size

1 3

4 5 6

DNA template PCR reaction

Product 1 Product 2
b) insertion or deletion of a small fragment of DNA - the
amplified fragments are changed in size

Small fragment DNA

Insertion 3
1 2
Deletion

4 5 6
DNA template
PCR reaction

Product 1 Product 2
c) insertion of a large piece of DNA between the primer -
binding sites may exceed the capacity of PCR - no fragment
is detected

The insertion of large fragment


2 3

5 6
DNA template PCR reaction

No product Product 2
A schematic picture of an agarose gel

Marker Plant A Plant B Plant C -

Monomorphic bands

Polymorphic bands

Presens of a band, ”1” Absence of a band, ”0”


And a real picture of a gel…
… and one more
 Most RAPD fragments result from the amplification of one locus, and two
kinds of polymorphism occur: the band may be present or absent, and the
brightness (intensity) of the band may be different (Figure 6)
Data analysis
A binary matrix
 RAPD bands are scored for present ”1” and absent ”0”.
Only clear, consistent and polymorphic bands are usually
used to create a binary matrix for future statistical analyses

Band 1 Band 2 Band 3 Band 4


Plant A 1 0 0 1
Plant B 0 1 0 1
Plant C 1 1 1 0
Plant D 1 1 0 1
Plant E 0 1 0 1
Plant F 1 0 0 1
Plant G 1 0 1 0
Statistical analyses
 Measurements of genetic diversity by means of
different genetic diversity indexes (i.e. Nei’s diversity
index, modified by Lynch and Milligan (1994) for
dominant markers, Shannon’s index etc)
 Cluster analysis, Multidimensional Scaling and
Principal co-ordinate analyses are used mainly for
evaluation of genetic relatedness among individual
organizms or among groups of organizms (i.e.
populations)
Keuntungan
 Tidak memerlukan pengetahuan sekuens DNA yang
dianalisis
 Distribusi acak di dalam genome
 Membutuhkan DNA dalam jumlah sedikit (5-20 ng)
 Mudah dan cepat pengerjaannya
 Efisien untuk mendapatkan marka dalam jumlah banyak
 Primer 10mer komersial dapat diterapkan untuk semua
spesies
 Menggunakan peralatan otomatis
 Biaya murah (Cost-effectiveness)!
Keterbatasan
 Marka dominan (individu heterozigot tidak dapat
dibedakan dari homozigot dominan)
 Sensitif terhadap perubahan kondisi reaksi yang
berpengaruh terhadap reproducibility pola pita
RAPD
 Interpretasi skoring pita RAPD
 Hasil tidak reproducible antara laboratorium
berbeda
Faktor yang berpengaruh terhadap
reproducibility reaksi RAPD:

 Kualitas dan kuantitas template DNA PCR buffer,


 Konsentrasi MgCl2,
 Rasio primer/template,
 Temperatur annealing,
 Merk atau sumber Taq DNA polymerase,
 Merk mesin PCR (thermal cycler)(Wolff et al.,
1993)
Aplikasi
 Keragaman genetik
 Struktur genetik populasi
 Karakterisasi plasma nutfah
 Verifikasi identitas genetik
 Pemetaan genetik
 Marka terpaut dengan karakter tertentu (trait of
interest)
 Identifikasi kultivar
 Identifikasi klon (variasi somaklonal)
 Interspecific hybridization
 Verifikasi kultivar dan kemurnian hibrid
 Klarifikasi tetua
The earlier identification of the seedless characteristic of the
wampee [Clausena lansium (Lour.) Skeels] hybrid by a random
amplified polymorphic DNA (RAPD) marker (Zhichang et al. 2010)
Genetic variation and population structure in Oryza
malampuzhaensis Krish. et Chand. endemic to Western Ghats,
South India (Thomas et al. 2001)