Genetic engineering is the process of identifying and isolating DNA from a living or dead cell and introducing it into another living cell. Before the genetic material is introduced, it may be altered in the laboratory. When the genetic engineering process is successful, the new DNA becomes permanently incorporated into the chromosomes of the new cell, and appears in the DNA of progeny cells as well. How can scientists do genetic engineering? They use recombinant DNA technology.
The methods developed for isolating, manipulating, amplifying, cutting and splicing together identifiable sequences of DNA are collectively called recombinant DNA technology.The next three sections will introduce several recombinant DNA techniques used to locate, isolate and amplify DNA. A final section shows how other recombinant techniques can be used to introduce new DNA into cells.
Many techniques in molecular biology are used to find out where a given gene is located within the human genome. This is not an easy task, since the human genome contains thousands of genes, many of which have not yet been found or sequenced. Of particular use in this task are DNA probes.
One way of finding a specific gene is through the use of a DNA probe, a relatively short single stranded DNA molecule that is complementary to a sequence on the gene of interest. In other words, if a segment of the gene of interest is known to be: AGTTCG, the complementary segment of the DNA probe would be TCAAGC, because A binds T and C binds G. (Actually, because of certain structural details about DNA molecules, the complementary sequence should actually be written as CGAACT, which is TCAAGC backwards. However, we are using the technically incorrect format here, to avoid confusion.)
An actual DNA probe would probably consist of at least a few dozen nucleotides complementary to a segment of the same length on the gene of interest. The probe is made to be radioactive, so that it can be detected easily.
Since a DNA probe will bind to single-stranded DNA, a technique called Southern Blotting can be performed which separates the double-stranded sample DNA into single strands and transfers them to a nylon membrane. When the probes are incubated with the membrane in a solution, they bind to complementary regions in the DNA and become "stuck" to the membrane. Afterwards, the membrane is put into contact with a sheet of photographic film that is sensitive to radioactive emission. Only those sections of the sample where the gene is located shows up as a dark spot on the paper, because these are the only sections bound to a radioactive probe.
DNA probes have several applications in molecular biology, including genetic methods of disease prediction and diagnosis , where probes that identify gene sequences known to be responsible for diseases are used. DNA probes can also be used in DNA fingerprinting , a technique often employed by forensic scientists to determine whether DNA found at a crime scene matches a suspect's DNA.
A DNA probe can be constructed even before the gene sequence itself is known! The way that this is done is by working backwards from the gene'sprotein product. Recall in our discussion of how proteins are made , we said that a gene is transcribed into messenger RNA (mRNA), based on simple rules of complementary nucleotide bases. The mRNA is then transported out of the nucleus and is used as a template for the assembly of a chain of amino acids, which folds into a protein.
We can isolate the protein produced by the gene we are interested in, and find out what the first 30 amino acids of the protein are. Based on this information, we can determine the first 90 nucleotides of that protein's mRNA template (recall that each sequence of 3 nucleotides codes for one amino acid, hence the 90:30 ratio). And because the mRNA template is complementary to the gene of interest, we now know what the sequence our DNA probe should be so that it is complementary to the first 90 nucleotides of the gene of interest.
To construct the DNA probe, we use a "Gene Machine," which is capable of synthesizing a short single-stranded DNA molecule containing the desired sequence of nucleotides in only a few hours.
If we were interested in introducing a human gene into another cell, it would not be sufficient to merely know that gene's location within the human genome. We would also need to isolate a copy of the gene, so that it can be inserted into the new cell.
For example, the human insulin gene must be isolated from human cells so that it can be incorporated into E. coli bacteria cells. The incorporated gene causes the bacterial cells to produce the human insulin protein, which can be administered to diabetics.
One method of isolating a gene is to work backwards from its protein product. First, at least part of the protein is sequenced, meaning that the order of the amino acids that make up the protein chain are determined. Usually, knowing the first 30 amino acids of the protein is enough. Next, based on the known amino acid sequence, and understanding the process of protein synthesis , we can predict the nucleotide sequence of part of the protein's mRNA template.
Next, a single stranded DNA probe is constructed that is complementary to the predicted mRNA sequence. For example, if part of the predicted mRNA sequence is CUA GUA CGA, the corresponding section of the DNA probe would be: GAT CAT GCT, because G' is complementary to C' and A' is complementary to T'. (Actually, because of certain structural details about DNA molecules, the complementary sequence should really be written as TCG TAC TAG, which is GAT CAT GCT backwards. However, we are using the technically incorrect format here, to avoid confusion.) The DNA probe is made to be radioactive so that it will be detectable when it binds to its DNA "mirror image."
The single-stranded DNA probe is then incubated with a sample thought to contain the complete mRNA protein template. When we isolate the mRNA to which the DNA probe binds, we have likely found the mRNA we are looking for.
Once the mRNA strand is found, all that must be done is to work backwards we need to synthesize the DNA strand that would have served as the template for the mRNA. That is, we need to synthesize DNA from RNA. There is an enzyme that allows us to do just that called reverse transcriptase, and which is found inside certain virus particles called retroviruses. These viruses employ RNA as their genetic material and use reverse transcriptase to generate DNA once they have infected a host cell. Biotechnologists can mix reverse transcriptase with mRNAs in vitro (outside of living cells in the laboratory, usually in a small plastic tube). As a result, the DNA sequence for the gene of interest is synthesized by the enzyme, based on the mRNA strand presented. Because the DNA produced was made artificially to be complementary to the mRNA, it is called a cDNA.
Often, DNA samples are in quantities that are too small to work with. Luckily, a technique invented in the 1980s called the Polymerase Chain Reaction (or PCR ), can be used to "amplify" the amounts of DNA in these samples.
The PCR machine is actually nothing more than a very precise heating and cooling device. The machine has small slots which hold small tubes containing the DNA sample and other necessary reaction ingredients. These additional ingredients include a generous supply of individual nucleotides (A's, T's C's and G's), short single-stranded DNA molecules called primers and an enzyme called Thermus aquaticus polymerase (Taq polymerase, for short). Taq polymerase is derived from bacteria that live in hot springs and are among the few enzymes that can function at very high temperatures.
The cycle begins when the machine heats the tube to a temperature somewhere around 90-95 C, pausing each double-stranded DNA molecule in the original sample to separate into two single stranded pieces.
(Recall that the hydrogen bonds that connect the two complementary strands of the DNA double helix are much weaker than the covalent bonds that connect the nucleotides together that make up each chain. The heating causes the hydrogen bonds to be broken, thus unravelling and separating the two strands, while the covalent bonds remain unaffected.)
Next, the temperature is lowered slightly, which allows the DNA primers to bind to the separated strands. The primers bind because they are complementary to certain sequences of each DNA strand which "flank" the DNA that is to be replicated in the middle. Once the primers have attached themselves to the single strands, Taq polymerase synthesizes, using the individual nucleotides floating around in the tube, a complementary strand for each original single strand. This completes the first cycle, and doubles the amount of sample DNA present in the tube.
In the next cycle, the PCR machine heats and cools as before, causing the new double-stranded DNA molecules to separate, and for new complementary strands to be synthesized by Taq polymerase.
Every time one cycle is performed, the amount of copies of the desired DNA sequence (located between the two primers) theoretically doubles. After about 30 cycles (which will typically take about 3 hours), enough DNA copies of this DNA sequence will have been made for the application of further biotechnology techniques.
The DNA Learning Centre, has developed an online video that gives a very good explanation of the PCR technique.
A gene, which is likely isolated in the form of a cDNA , can be introduced into a cell using a vector. A vector is a vehicle by which foreign DNA is transferred from one cell to another.
Some examples of vectors include modified viruses and plasmids.
Viruses are excellent vectors, because they have gained, through long periods of evolution, the ability to avoid destruction by the human immune system, and have the capacity to get their own genetic material inside specific cells. As we examined in the section describing viruses, a viral infection consists of foreign (viral) genetic material entering the cell and harnessing the cell's nucleic acid and protein-making machinery to produce its own DNA, RNA and proteins. To use a virus as a vector, the harmful sections of its DNA are replaced with the desired cDNA to be introduced in the cell. Then, we allow the virus to infect' our host cell and if all goes well, the cDNA enters the cell and will be used to make the desired protein.
Some viruses can produce their own DNA and incorporate it into the host cell's genome. These RNA-based retroviruses are the most common viral vectors used in Gene Therapy, where genes with therapeutic value are inserted into the retroviruses which, upon infection, incorporate these into the genome of the recipient cell.
It should be noted that viruses that are to be used as vectors are made to be "replication defective". In other words, the harmful parts of the viral genome that serve to produce more viral particles have been removed and replaced by a sequence that codes for the protein of interest.
The way in which the human insulin cDNA is introduced into bacterial cells is through the use of a plasmid. A plasmid is simply a loop of DNA containing genes that can easily diffuse into and out of bacterial cells. Although plasmids occur naturally in certain bacteria, the plasmids used for the purpose of introducing and expressing a foreign gene into a cell have been altered to such an extent, that the sequences they contain are very different from the naturally occurring plasmids upon which they are based.
For one thing, the plasmid contains several specialized short sequences called restriction sites. Enzymes called restriction endonucleases recognize these sites and cut the plasmid DNA. For example, a restriction enzyme called EcoR1 recognizes the sequence GAATTC and cuts between the G and the first A. Notice that the complementary sequence is CTTAAG, which is GAATTC backwards! So the enzyme cuts both strands of the plasmid like so:
Notice that cutting using EcoR1 generates two "sticky ends" which are single stranded strings of nucleotides that will bind to a complementary set of single-stranded "sticky ends." Most importantly, the plasmid is engineered so that this particular restriction sequence is present only once, meaning that EcoR1 will cut the double-stranded plasmid at only one location.
The cDNA (which contains the human insulin gene) to be inserted into the plasmid is altered depending on the restriction enzyme that was used to cut the plasmid. In keeping with our Eco R1 example, the following "sticky ends" would be needed on each end of the cDNA:
Now we incubate the altered cDNA sequence in a solution with a plasmid cut using the EcoR1 enzyme and with an enzyme that fastens DNA pieces together (called DNA ligase). This results in a closed circular plasmid which contains the cDNA, and therefore the human insulin gene.
The plasmid is subsequently incubated with bacterial cells (in the case of the insulin process, the bacteria used are a species called E. coli) under specific conditions which favour the absorption of the plasmid by the bacterial cell.
In theory, the plasmid containing the human insulin gene will enter all of the bacterial cells and all of these cells will transcribe the protein and produce human insulin, which can then be harvested and used to treat diabetic patients.
Unfortunately, not all the bacteria cells will actually absorb the plasmid. In fact, in most cases, relatively few of them will absorb it. How can biotechnologists select only those bacteria cells that have absorbed the plasmid?
The answer lies in the bacterial culture conditions and in another special modification built into genetically engineered plasmids. The bacteria, after they have been incubated in the presence of the plasmid (and some have absorbed it), are cultured in a medium that contains an antibiotic, like ampicillin. Ampicillin will kill E. Coli bacteria, unless they are protected in some way. The plasmid that the bacteria have absorbed also contains a gene that confers a resistance to ampicillin. Therefore, only those bacteria that have absorbed the plasmid will be resistant to the antibiotic and will survive. Since the plasmid also contains the gene for human insulin, we have allowed only those bacteria that may be capable of producing insulin to survive and multiply.