Teknologi DNA rekombinan (rekayasa genetika, genetic engineering) telah digunakan untuk mengisolasi dan memanipulasi gen-gen secara in vitro guna memberikan kemampuan sintesis baru pada sel.

Produk yang dihasilkan melalui teknik rekayasa genetika misalnya:

1. Faktor pembekuan darahseperti aktivator plasminogen jaringan yang digunakan untuk mengaktivasi penghancuran bekuan darah dan mencegah terjadinya bekuan darah lagi pada pasien serangan jantung.

2. komponen-komponen komplemen (bagian dari sistem imun)

3. Pada bidang agrikultur: kedelai, jagung, dan kapas telah direkayasa secara genetik (dimodifikasi) agar mengandung gen tambahan yang tahan terhadap herbisida dan hama. Tanaman itu disebut Genetically Modified Organism (GMO).

Dll...

PCR ditemukan oleh KARY MULLIS pada tahun 1985.

What is PCR?PCR dilakukan di dalam tabung reaksi (eppendorf) melalui pencampuran DNA dengan 1 set reagen dan kemudian ditempatkan dalam mesin thermal cycler (mesin yang memungkinkan inkubasi campuran larutan pada suatu seri temperatur yang bervariasi melibatkan 30 atau 40 siklus reaksi).

The polymerase chain reaction (PCR) is a technique by which small samples of DNA can be quickly amplified, that is, increased to quantities that are la rge enough for analysis.

Starting with just one gene-sized piece of DNA, PCR can be used to make literally billions of copies in only a few hours.

Tujuan: mendapatkan sejumlah besar salinan suatu gen dalam waktu singkat.

UNTUK MENJALANKAN REAKSI PCR DIBUTUHKAN:

1. Molekul DNA (template); tidak harus murni, dapat diperoleh dari genomic DNA, fosil, sampel forensik (darah, cairan semen, potongan rambut), dll.

2. PCR primer; merupakan potongan pendek DNA (biasanya 10-30 nukleotida) yang akan mengapit daerah target yang hendak disalin sehingga dapat dimulai sintesa DNA baru.

3. Enzim (Taq polymerase)4. Nukleotida5. Mesin thermocycler PCR

3 tahap reaksi PCR pada tiap siklus:

1. DENATURASI; tahap pendenaturasian DNA untai ganda menjadi untai tunggal dengan pemanasan hingga 90-95oC selama sekitar 5menit.

2. ANNEALING; suhu reaksi diturunkan hingga 50-70oC sehingga primer dapat berikatan dengan DNA yang telah terdenaturasi.

3. EKSTENSI; tahap sintesa DNA baru (70-75oC). Taq polimerase akan memulai reaksi pemanjangan rangkaian primer dengan adanya nuleotida pada arah 5’-3’.

PCR is a chain reaction because the number of new DNA strands is doubled in each cycle, and the new strands, along with the old strands, serve as templates in the next cycle (Klug and Cummings, 2005).

For the many different applications of the PCR, many different methods of isolating and preparing the template DNA exist, mostly depending on the source of the DNA.

It is important to note that the outcome of a PCR is dependent on the quality and integrity of the template DNA.

It is wise to purify template DNA using a product or method that is specifically designed to purify template DNA for use in PCR

Generally, the amount of DNA per reaction should be 104-106 target/template molecules.

DNA template

Three steps of Polymerase Chain Reaction (Adopted from Klug and Cummings)

electrophoresis didasarkan pada perbedaan kecepatan gerak protein

dalam medan listrik pada pH tertentu.

Detection of the PCR product or “amplicon” can be accomplished several ways.

Following PCR, the sample is loaded into an agarose gel, and the DNA fragment(s) or amplicon if present in the sample is separated by electrophoresis based on size.

Molecular weight, DNA standards are included to estimate size of amplicon(s) present in positive samples and positive control.

The agarose gel and electrophoresis buffer contain a dye, ethidium bromide that binds double stranded DNA and fluoresces upon excitation with UV light.

This dye is used to visualize the DNA in an agarose gel.

Analysis of PCR product“Agarose gel electrophoresis”

There is an inverse linear correlation between the log10 size of the DNA fragment (bp) and the distance migrated by the DNA fragment in the agarose gel.

The smaller the DNA fragment, the farther it migrates through the agarose gel during electrophoresis.

As most PCRs produce small size amplicons (100–1,000 bp), one must use DNA standards that accommodate this size range and agarose concentration (1.5%) that resolves small DNAfragments sufficiently to accurately determine the size for DNA band X.

The PCR result is recorded photographically with a polaorid or digital camera with the appropriate lens filters and exposures for capturing images illuminated by the UV light.

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 M

0.25

0.50.751.01.5

2.03.0

10.0

kb

Figure 7.2. RAPD fingerprints obtained using primer OPAR3 for K. pneumoniae and E. cloacae. Lane M: 1 kb molecular marker. Lane 18-K12; 19-K13; 20-K14; 21-K15; 23-K16; 24-K17; 25-K18; 27-K19; 28-K20; 29-K21; 30-K22; 31-K23; 33-K24; 34-K25 (K = K. pneumoniae) Lane 22-E7 (E = E. cloacae).