Teknologi DNA Rekombinan

n  Dengan ditemukannya DNA, manusia berupaya untuk mendapatkan kombinasi sifat-sifat baru suatu mahluk hidup dengan cara melakukan perubahan langsung pada DNA genomnya.

n  Usaha untuk mengubah DNA genom secara langsung ini disebut dengan istilah Rekayasa Genetika atau Genetic Engineering.

n  Dalam upaya melakukan rekayasa genetika, manusia menggunakan teknologi DNA rekombinan.

Teknologi DNA rekombinan

n  Teknologi DNA Rekombinan merupakan kumpulan teknik atau metoda yang digunakan untuk mengkombinasikan gen-gen

n  Teknologi DNA rekombinan telah mungkinkan bagi kita untuk: mengisolasi DNA dari berbagai organisme, menggabungkan DNA yang berasal dari organisme yang berbeda sehingga terbentuk DNA rekombinan, memasukkan DNA rekombinan ke dalam sel organisme prokariot maupun eukariot hingga DNA rekombinan dapat berepilkasi dan bahkan dapat diekspresikan.

n  Teknik-teknik tersebut meliputi:

1.      Teknik untuk mengisolasi DNA.

2.      Teknik untuk memotong DNA.

3.      Teknik untuk menggabung atau menyambung DNA.

4.      Teknik untuk memasukkan DNA ke dalam sel hidup.

Berbagai teknik yang digunakan dalam teknologi DNA Rekombinan

1.           Complementary DNA

Complementary DNA (cDNA) adalah DNA yang dibuat berdasarkan sekuen mRNA.

Pembuatannya melalui pemanfaatan enzim reverse transcriptase, yang melakukan kebalikan dari transkripsi,yaitu sintesis DNA dari templat RNA.

Enzim ini dihasilkan secara alami oleh kelompok virus yang disebut retroviruses (termasuk virus HIV), dan enzim ini membantu dalam menginvasi sel-sel.

Dalam rekayasa genetika enzim ini digunakan untuk membuat gen artifisial, cDNA. 

cDNA has helped to solve different problems in genetic engineering:

n  It makes genes much easier to find. There are some 70 000 genes in the human genome, and finding one gene out of this many is a very difficult (though not impossible) task.

n  However a given cell only expresses a few genes, so only makes a few different kinds of mRNA molecule.

n  For example the b cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene.

2.           Restriction Enzymes 

These are enzymes that cut DNA at specific sites.

They are properly called restriction endonucleases because they cut the bonds in the middle of the polynucleotide chain.

Their biochemical activity is the hydrolysis ("digestion") of the phosphodiester backbone at specific sites in a DNA sequence.

Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes make a staggered cut in the two strands, forming sticky ends.

The cut ends are “sticky” because they have short stretches of single-stranded DNA with complementary sequences. These sticky ends will stick (or anneal) to another piece of DNA by complementary base pairing, but only if they have both been cut with the same restriction enzyme.

n  Enzim restriksi biasanya diisolasi dari bakteri

n  Yang digunakan dalam rekayasa genetika: enzim restriksi endonuklease, enzim ini mengenali DNA pada situs khusus dan memotong pada situs tsb.

n  Situs pengenalan ER adalah daerah yg simetri atau disebut dg palindrom, artinya bila kedua utas DNA tsb masing-masing dibaca dg arah yg sama akan memberikan urutan nukleotida yg sama.

n   Pemotongan ER menghasilkan dua jenis ujung potongan: berujung rata/ blunt end, dan berujung tidak rata/sticky end/ cohesive. ER yg memotong pada pusat palindrom akan menghasilkan potongan berujung rata, sedangkan ER yang memotong diluar pusat simetri menghasilkan potongan berujung kohesif

n  Restriction enzymes are highly specific, and will only cut DNA at specific base sequences,  4-8 base pairs long, called recognition sequences.

n  Restriction enzymes are produced naturally by bacteria as a defence against viruses (they “restrict” viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called restriction fragments. There are thousands of different restriction enzymes known, with over a hundred different recognition sequences.

n  Restriction enzymes are named after the bacteria species they came from, so EcoR1 is from E. coli strain R, HindIII is from Haemophilis influenzae.

3.           DNA Ligase

Enzim ligase menyambung dua ujung DNA melalui ikatan kovalen antara ujung 3’OH dari utas yang satu dengan ujung 5’P dari utas yang lain.

2 tipe enzim ligase yg sering digunakan: DNA ligase dari E.coli, dan DNA ligase dari fage T4.

Ujung kohesif hanya dpt disambung dg ujung kohesif yg kompatibel dan memenuhi komplementaritas (A-T dan G-C). Ujung kohesif lebih efisien dlm penyambungan. 

This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments.

The sticky ends allow two complementary restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete.

DNA ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources.  

4.           Vectors 

In biology a vector is something that carries things between species. For example the mosquito is a disease vector because it carries the malaria parasite into humans.

In genetic engineering a vector is a length of DNA that carries the gene we want into a host cell.

A vector is needed because a length of DNA containing a gene on its own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty quickly.

A vector gets round these problems by having these properties:

n  It is big enough to hold the gene we want, but not too big.

n  It is circular (a closed loop), so that it is less likely to be broken down (particularly in prokaryotic cells where DNA is always circular).

n  It contains control sequences, such as a replication origin and a transcription promoter, so that the gene will be replicated, expressed, or incorporated into the cell’s normal genome.

n  It contain marker genes, so that cells containing the vector can be identified.

n  Many different vectors have been made for different purposes in genetic engineering by modifying naturally-occurring DNA molecules

n  For example a cloning vector contains sequences that cause the gene to be copied (perhaps many times) inside a cell, but not expressed. An expression vector contains sequences causing the gene to be expressed inside a cell, preferably in response to an external stimulus, such as a particular chemical in the medium.

5.           Plasmids 

Plasmids are by far the most common kind of vector, so we shall look at how they are used in some detail.

Plasmids are short circular DNA found naturally in bacterial cells.

A typical plasmid contains 3-5 genes and there are usually around 10 copies of a plasmid in a bacterial cell.

Plasmids are copied separately from the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells.

They are also used naturally for exchange of genes between bacterial cells (the nearest they get to sex), so bacterial cells will readily take up a plasmid.

Because they are so small, they are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.

n  One of the most common plasmids used is the R-plasmid (or pBR322).

n  This plasmid contains a replication origin, several recognition sequences for different restriction enzymes (with names like PstI and EcoRI), and two marker genes, which confer resistance to different antibiotics (ampicillin and tetracycline).  

n  The restriction enzyme used here (PstI) cuts the plasmid in the middle of one of the marker genes (we’ll see why this is useful later). The foreign DNA anneals with the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA).  Several other products are also formed: some plasmids will simply re-anneal with themselves to re-form the original plasmid, and some DNA fragments will join together to form chains or circles. Theses different products cannot easily be separated, but it doesn’t matter, as the marker genes can be used later to identify the correct hybrid vector.

n  The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction and ligase enzymes.

   6. Polymerase Chain Reaction (PCR)

•         Genes can be cloned by cloning the bacterial cells that contain them, but this requires quite a lot of DNA in the first place.

•         PCR can clone (or amplify) DNA samples as small as a single molecule.

•         It is a newer technique, having been developed in 1983 by Kary Mullis, for which discovery he won the Nobel prize in 1993. T

•         he polymerase chain reaction is simply DNA replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated many times.

1.      Start with a sample of the DNA to be amplified, and add the four nucleotides and the enzyme DNA polymerase.

2.      Normally (in vivo) the DNA double helix would be separated by the enzyme helicase, but in PCR (in vitro) the strands are separated by heating to 95°C for two minutes. This breaks the hydrogen bonds.

3. DNA polymerisation always requires short lengths of DNA (about 20 bp long) called primers, to get it started.

•         In vivo the primers are made during replication by DNA polymerase, but in vitro they must be synthesised separately and added at this stage.

•         This means that a short length of the sequence of the DNA must already be known, but it does have the advantage that only the part between the primer sequences is replicated. The DNA must be cooled to about 40°C to allow the primers to anneal to their complementary sequences on the separated DNA strands.

4. The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of the DNA.

•         The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90°C, so it is not denatured by the high temperatures in step 2. Its optimum temperature is about 72°C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible.

5. Each original DNA molecule has now been replicated to form two molecules.

•         The cycle is repeated from step 2 and each time the number of DNA molecules doubles.

•          This is why it is called a chain reaction, since the number of molecules increases exponentially, like an explosive chain reaction. Typically PCR is run for 20-30 cycles.

6. PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified millions of times with little effort.

•         The product can be used for further studies, such as cloning, electrophoresis, or gene probes.

•         Because PCR can use such small samples it can be used in forensic medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy DNA from mummified human bodies, extinct woolly mammoths, or from an insect that's been encased in amber since the Jurassic period.  

7.Gene Transfer

•         Vectors containing the genes we want must be incorporated into living cells so that they can be replicated or expressed. The cells receiving the vector are called host cells, and once they have successfully incorporated the vector they are said to be transformed. Vectors are large molecules which do not readily cross cell membranes, so the membranes must be made permeable in some way. There are different ways of doing this depending on the type of host cell.

•         Heat Shock. Cells are incubated with the vector in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the vector, though no one knows why. This works well for bacterial and animal cells.

•         Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell. This is the most efficient method of delivering genes to bacterial cells.

•         Viruses. The vector is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material. Since viruses rely on getting their DNA into host cells for their survival they have evolved many successful methods, and so are an obvious choice for gene delivery. The virus must first be genetically engineered to make it safe, so that it can’t reproduce itself or make toxins. Three viruses are commonly used:

1.         Bacteriophages (or phages) are viruses that infect bacteria. They are a very effective way of delivering large genes into bacteria cells in culture.

2.         Adenoviruses are human viruses that causes respiratory diseases including the common cold. Their genetic material is double-stranded DNA, and they are ideal for delivering genes to living patients in gene therapy. Their DNA is not incorporated into the host’s chromosomes, so it is not replicated, but their genes are expressed.

•         The adenovirus is genetically altered so that its coat proteins are not synthesised, so new virus particles cannot be assembled and the host cell is not killed.

3.         Retroviruses are a group of human viruses that include HIV. They are enclosed in a lipid membrane and their genetic material is double-stranded RNA. On infection this RNA is copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the foreign genes are replicated into every daughter cell.

•         After a certain time, the dormant DNA is switched on, and the genes are expressed in all the host cells.

•         Plant Tumours. This method has been used successfully to transform plant cells, which are perhaps the hardest to do. The gene is first inserted into the Ti plasmid of the soil bacterium Agrobacterium tumefaciens, and then plants are infected with the bacterium. The bacterium inserts the Ti plasmid into the plant cells' chromosomal DNA and causes a "crown gall" tumour. These tumour cells can be cultured in the laboratory and whole new plants grown from them by micropropagation. Every cell of these plants contains the foreign gene.

•         Gene Gun. This extraordinary technique fires microscopic gold particles coated with the foreign DNA at the cells using a compressed air gun. It is designed to overcome the problem of the strong cell wall in plant tissue, since the particles can penetrate the cell wall and the cell and nuclear membranes, and deliver the DNA to the nucleus, where it is sometimes expressed.

•         Micro-Injection. A cell is held on a pipette under a microscope and the foreign DNA is injected directly into the nucleus using an incredibly fine micro-pipette. This method is used where there are only a very few cells available, such as fertilised animal egg cells. In the rare successful cases the fertilised egg is implanted into the uterus of a surrogate mother and it will develop into a normal animal, with the DNA incorporated into the chromosomes of every cell.

8. Genetic Markers

•         These are needed to identify cells that have successfully taken up a vector and so become transformed. With most of the techniques above less than 1% of the cells actually take up the vector, so a marker is needed to distinguish these cells from all the others. We’ll look at how to do this with bacterial host cells, as that’s the most common technique.

•         A common marker, used in the R-plasmid, is a gene for resistance to an antibiotic such as tetracycline. Bacterial cells taking up this plasmid can make this gene product and so are resistant to this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal untransformed cells, together with cells that have taken up DNA that’s not in a plasmid (99%) will die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate.

9.    DNA Probes

•         These are used to identify and label DNA fragments that contain a specific sequence. A probe is simply a short length of DNA (20-100 nucleotides long) with a label attached. There are two common types of label used:

–     a radioactively-labelled probe (synthesised using the isotope 32P) can be visualised using photographic film (an autoradiograph).

–     a fluorescently-labelled probe will emit visible light when illuminated with invisible ultraviolet light. Probes can be made to fluoresce with different colours.

•         Probes are always single-stranded, and can be made of DNA or RNA. If a probe is added to a mixture of different pieces of DNA (e.g. restriction fragments) it will anneal (base pair) with any lengths of DNA containing the complementary sequence. These fragments will now be labelled and will stand out from the rest of the DNA.

DNA probes have many uses in genetic engineering:

•         To identify restriction fragments containing a particular gene out of the thousands of restriction fragments formed from a genomic library. This use is described in shotguning below.

•         To identify the short DNA sequences used in DNA fingerprinting.

•         To identify genes from one species that are similar to those of another species. Most genes are remarkably similar in sequence from one species to another, so for example a gene probe for a mouse gene will probably anneal with the same gene from a human. This has aided the identification of human genes.

•         To identify genetic defects. DNA probes have been prepared that match the sequences of many human genetic disease genes such as muscular dystrophy, and cystic fibrosis. Hundreds of these probes can be stuck to a glass slide in a grid pattern, forming a DNA microarray (or DNA chip). A sample of human DNA is added to the array and any sequences that match any of the various probes will stick to the array and be labelled. This allows rapid testing for a large number of genetic defects at a time.

10.   Antisense Genes

•         These are used to turn off the expression of a gene in a cell. The principle is very simple: a copy of the gene to be switch off is inserted into the host genome the “wrong” way round, so that the complementary (or antisense) strand is transcribed. The antisense mRNA produced will anneal to the normal sense mRNA forming double-stranded RNA. Ribosomes can’t bind to this, so the mRNA is not translated, and the gene is effectively “switched off”.

11.    Gene Synthesis

•         It is possible to chemically synthesise a gene in the lab by laboriously joining nucleotides together in the correct order.  Automated machines can now make this much easier, but only up to a limit of about 30bp, so very few real genes could be made this way (anyway it’s usually much easier to make cDNA). The genes for the two insulin chains (xx bp) and for the hormone somatostatin (42 bp) have been synthesisied this way. It is very useful for making gene probes.

•         12.    Electrophoresis This is a form of chromatography used to separate different pieces of DNA on the basis of their length. It might typically be used to separate restriction fragments. The DNA samples are placed into wells at one end of a thin slab of gel made of agarose or polyacrylamide, and covered in a buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive electrode). The molecules have to diffuse through the gel, and smaller lengths of DNA move faster than larger lengths, which are retarded by the gel. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time. At the end of the run the current is turned off.

•         Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are three common methods for doing this:

•          The gel can be stained with a chemical that specifically stains DNA, such as ethidium bromide or azure A. The DNA shows up as blue bands. This method is simple but not very sensitive.  

•         The DNA samples at the beginning can be radiolabelled with a radioactive isotope such as 32P. Photographic film is placed on top of the finished gel in the dark, and the DNA shows up as dark bands on the film. This method is extremely sensitive.

•         The DNA fragments at the beginning can be labelled with a fluorescent molecule. The DNA fragments show up as coloured lights when the finished gel is illuminated with invisible ultraviolet light.