presentation rapd

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 technologyA B C

Genomic DNA

+

Taq polymerase

+

Arbitrary primers

A

+

Nucleotides

+

Buffer

PCR

(under relaxed conditions)

Electrophoresis

PCR

360 bp

260 bp

520 bp

520bp

260 bp

360 bp

A B C

A B C

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

5 6

1 2 3

4

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

5

1 3

4 6

Product 2

PCR reaction

2

DNA template

No product

or detected fragment is of increased size

2

PCR reaction

1 3

4 6

Product 2

DNA template

5

Product 1

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

2 3

5

DNA templatePCR reaction

6

Product 1 Product 2

Small fragment DNA

Insertion

Deletion1

4

2 3

5DNA template PCR reaction

6

Product 2No product

The insertion of large fragment

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

A schematic picture of an agarose gel

Plant AMarker 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

Komposisi reaksi PCR

1X larutan penyangga (50 mM KCl, 10 mM Tris-HCl pH 9, 0,01% Triton X-100),

MgCl2 (1.5-10 mM)

dNTP Primer (0.1-2.0 mM) Taq polimerase DNA DNA genom (10-50 ng/50µl volume reaksi)

Arbitrary Primer

The RAPD protocol usually uses a 10 bp arbitrary primer at constant low annealing temperature (generally 34 – 37 oC).

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)

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

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

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

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)