The Biology Topics of biotechnology involve using living organisms to develop new products or solve problems.
Basic Techniques to Manipulate Genetic Material – Overview of Genetic Modification Techniques
Various techniques are used in genetic engineering. Several such techniques may be elaborated as follows:
A. Isolation of DNA
Isolation of the desired gene from the genome of an organism and its cloning is necessary in genetic engineering. In the first phase, the desired gene is isolated and inserted into a plasmid vector to produce a recombinant vector. The vector then carries the gene to the host for its cloning. Therefore, isolation of a gene is the primary requirement for genetic engineering. Total genomic DNA isolation is often carried out according to the following procedure.
The host cell wall is lysed. The method used for the lysis procedure depends upon the nature of the host cell itself. For instance, a culture of bacteria is grown and then harvested. Bacterial cells are often treated with the enzyme lysozyme to weaken their cell wall before being lysed with detergents. Yeast cells, on the other hand, are treated with zymolase to disrupt the integrity of their cell wall before lysis proceeds often by grinding the cells using glass beads to break them open. Cellulase (for plant cells) and chitinase (for fungus) are also used.
This cell extract is treated to remove all components except the DNA. The cellular debris will be removed by centrifugation. The remaining soluble material will then be vortexed in the presence of phenol to remove proteins by denaturing them. Chloroform is often used in conjunction with phenol (as a phenol/chloroform solution) since it is also a protein denaturant, but it also stabilizes the rather unstable boundary between the aqueous phase and a pure phenol layer. Proteins can also be removed by treatment with protease.
The nucleic acid remaining in the aqueous layer can then be precipitated using ethanol. Upon addition of ethanol, the DNA will form a stringy white precipitate that can be collected by centrifugation. RNA can be removed from the preparation by treatment with RNase.
B. Methods of Selection of the Recombinants
For cloning a recombinant containing the rDNA must be selected. Therefore, it is essential to identify a host cell that has a recombined DNA. The method by which a recombinant is screened is known as selection. There are various methods for such selection. However, the three methods are straightforward and they may be discussed below.
(i) Insertional inactivation
This technique is more appropriate for the selection of the recombinant host (bacteria). In this approach, one of the genetic traits is disrupted so that expression of the disrupted gene does not occur.
On observing the inactivation of a gene, the recombined bacterium may be recognized. In plasmid, there are two antibiotic resistance genes namely ampR and tetR.(Ampicillin resistance and tetracyclin resistance). With the use of BamHI when pBR322 is used for gene insertion the foreign DNA is inserted within tetR gene. In that case, the tetR gene becomes inactivated. Therefore, whether the foreign DNA is inserted or not can be identified as inactivation of the tetR gene. The bacteria containing the recombined plasmid fail to grow in the medium containing both ampicillin and tetracyclin as antibiotics. On the other hand, the recombined host bacteria grow in the medium containing only ampicillin.
(ii) Blue-White Selection
The technique is more efficient in the selection of recombinants. One plasmid is first made recombined with the insertion of lac Z. Lac Z is called the reporter gene and the gene may produce β-galatosidase which produces blue colour by reacting with a synthetic substrate X-gal.
In an experiment, a foreign DNA is inserted within the lac Z gene. It is to be mentioned here that lac Z has several recognition sites for restriction enzymes. Because of the insertion of foreign DNA within Lac Z, the recombinant fails to produce β-galactosidase. In another experiment, foreign DNA is inserted outside the lac Z gene, and in this case, β-galactoside production cannot be prevented. Consequently, some should not get any foreign DNA except the reporter gene.
Now these three forms of bacteria are grown in a culture plate with a medium containing X-gal, two types of colonies, blue-coloured colonies and white colonies. From this it can easily be recognized which recombinants contain foreign genes within lac Z. This by blue-white selection the recombinants for cloning may be selected. It is important to note that the recombinant plasmids are inserted in the host bacteria before their culture in X-gal-containing media on the culture plates.
(iii) Antibiotic Resistance
In a bacterial host, a plasmid vector is normally employed for gene transfer. The plasmid vector contains a marker gene as antibiotic resistance for easy recognition. If a plasmid contains a gene for ampicillin resistance (ampr) and it is used for foreign gene insertion, the bacterial host containing such plasmid will be a recombinant. A medium containing ampicillin allows only the recombined bacteria as they contain the gene for ampicillin resistance. The from a group of bacteria the recombinants may directly be selected if they are cultured in a medium containing ampicillin.
N.B.: Selection involves the application of some short of pressure for example the presence of antibiotics during the growth of host cells containing the recombinant DNA. The cells with desired characteristics are selected by their survival ability.
C. Formation of Recombinant DNA
The desired DNA is usually smaller in comparison to donor DNA. Therefore, with the help of restriction endonuclease the donor DNA is fragmented. From the fragments of DNA, the required DNA fragment (the gene itself) can be recognized with the help of a radioactive probe. A probe is a defined and radioactively or non-radioactively labeled nucleic acid sequence used in molecular cloning to identify specific DNA molecules with complementary sequence(s). by auto radiography or with non-radioactive DNA-detection systems. After the identification of the desired or target gene is done, the DNA fragment may be combined with a vector DNA to obtain a recombinant DNA molecule.
For this purpose, each vector molecule must be cleaved at a single position to open up the circle so that new DNA can be inserted. The vector was cut by the same restriction endonuclease that was used to cut the gene of interest so that both the vector and the gene of interest contain the same sticky ends. Then DNA ligase is used to join the vector and the gene of interest together to generate a recombinant vector with the desired gene inserted into it.
D. Method for Insertion of a Gene or DNA into Host Cell
For introduction of a recombinant vector carrying the desired gene or DNA fragment into the host cell is necessary for in vivo cloning of a gene. When their recombinant vector containing desired gene is introduced in the cell, the vector may be replicated many times to produce many copies of the desired gene (called in vivo gene cloning) or the desired gene inserted into the recombinant vector may produce proteins after transcription and translation. For the introduction of the recombinant vector with the inserted desired gene into the host cell several methods are used transformation, electroporation, and lipofection.
Transformation is a natural process and in this process, a foreign DNA may be taken up by the bacterial cell. If the DNA fragment is small the bacterial cell may take it from the medium through its cell membrane. Most species of bacteria, including E. coli, take up only limited amounts of DNA under normal circumstances. In order to transform these species efficiently, the bacteria have to undergo some form of physical and/or chemical treatment that enhances their ability to take up DNA. Cells that have undergone this treatment are said to be competent. If bacterial cells are soaked in an ice-cold 50 mM calcium chloride (CaCl2) solution they efficiently take up exogenous DNA. The DNA may adhere to the surface of the cell and uptake is mediated by a pulsed heat shock (30 seconds) at 37-450 C.
Electroporation is the use of an electric field pulse to induce microscopic pores within a biological membrane. These pores, called ‘electrophoresed’, allow molecules, ions, and water to pass from one side of the membrane to the other. If a suitable electric field pulse is applied [short (1 msec) electric pulse and potential gradients of 700V/cm], then the electroporated cells can recover, with the electrophoresis resealing spontaneously, and the cells can continue to grow. Pore formation is extremely rapid (approximately 1 ps), while pore resealing is much slower, and is measured in the order of minutes. In this method, a gene may be introduced in the bacterial cell with the help of high-voltage of electric shock. The electric shock increases the permeability of the cell membrane of bacteria and then the bacteria may take up a piece of DNA from the medium through its cell membrane. DNA can enter the bacterial cell before the pores spontaneously reseal.
Transfection is the introduction of foreign DNA into the genome of cultured animal or human cells via direct gene transfer (e.g., by calcium phosphate precipitation). The stable transfection leads to the incorporation of the foreign DNA into the genome of the recipient cell, whereas transient transfection restricts the DNA to the cytoplasm, where it is rapidly destroyed, i.e., the encoded genes are only transiently expressed and not integrated into the host cell genome.
(iii) Gene Gun
The gene gun or ‘Biolistic gun’ is a device that literally fires DNA into target cells. The DNA to be transformed into the cells is coated onto microscopic beads made of either gold or tungsten. The coated beads are then attached to the end of a plastic bullet and loaded into the firing chamber of the gene gun. An explosive force fires the bullet down the barrel of the gun towards the target cells that lie just beyond the end of the barrel.
When the bullet reaches the end of the barrel it is caught and stopped, but the DNA-coated beads continue on towards the target cells. Some of the beads pass through the cell wall and into the cytoplasm of the target cells. Here, the bead and the DNA dissociate and the cells become transformed. The gene gun is particularly useful for transforming cells that are difficult to transform by other methods, e.g., plant cells. It is also gaining in use as a method for transferring DNA constructs into whole animals.
(iv) Liposome-mediated gene transfer
A liposome is a type of fluid-filled lipid sac. When this sac comes in contact with the cell membrane it may be fused with the cell. Therefore, by introducing a DNA fragment into the liposome and mixing the liposome with the host cell, the DNA fragments may be introduced into the cell. This procedure of gene transfer is called lipofection.
Lipofection (liposome-mediated gene transfer):
A liposome is an artificially generated lipid or phospholipid vesicle of about 25nm to 1μm in diameter, consisting of a lipid bilayer enclosing a single aqueous compartment. Lipofection is a simple and effective direct gene transfer technique to introduce up to 120kb of DNA into eukaryotic cells by entrapping them into small liposomes consisting of synthetic cationic lipids.
DNA can be entrapped in such liposomes by simply mixing phospholipids (e.g., phosphatidylserine) and buffer containing DNA by brief sonication. Loaded vesicles are then fused with membranes of recipients (in the case of plant cells the plasma membranes are only accessible after en¬zymatic removal of the cellulose cell wall) and deliver the DNA into the cell.
(v) Calcium Phosphate Precipitation
It is a method to introduce DNA into a target cell without a vector (vector-less gene transfer, direct gene transfer) that is based on the precipitation of the DNA in insoluble calcium phosphate complexes directly onto the membranes of the cell. These calcium phosphate precipitates are generated after adding a DNA-CaCl2 solution to an isotonic phosphate buffer. The precipitates that form after about 30 minutes are used to transform bacterial, plant protoplasts, and animal cells with high efficiency.
(vi) Microinjection of naked DNA
This process makes use of a very fine pipette to inject DNA molecules directly into the nucleus of the cells to be transformed and also uses a micromanipulator consisting of a glass capillary with a blunt end (“holding capillary”) to position the recipient cell and to hold it under slight suction. This technique was initially applied to animal cells but has subsequently been successful with plant cells.
E. Cloning of DNA
The procedure by which many copies of a DNA fragment may be produced is known as DNA cloning. The newly made copies of the same DNA molecule are called clones of the original DNA. To know the composition of a gene and its function cloning is much helpful. In some situations, where a very small quantity of DNA sample could be isolated, DNA cloning becomes essential to produce large quantities of DNA fragments which are employed to various other techniques such as DNA fingerprinting and DNA blotting.
By introducing a recombinant vector containing the desired gene or DNA fragment into the host bacterial cell or by polymerase chain reaction the cloning of a DNA sequence may be achieved. The first method is called in vitro DNA cloning and the second method of DNA cloning is called cell-based (in vivo) DNA cloning.
(i) In vitro gene cloning by PCR
With the help of a PCR machine in vitro gene cloning is done. PCR machine(also called thermocycler) was discovered in 1983 by Karry Mullis. With the help of a particular DNA polymerase called Taq polymerase the DNA in the machine is replicated at higher temperatures. Taq polymerase is obtained from Thermus aquaticus a bacterial species that lives in hot spring and the bacteria use this enzyme to replicate their DNA at high temperatures. The other ingredients required for PCR are:
- Template DNA (the sample to be copied is called the template DNA. Often this is a known sequence or gene, typically double-stranded, and they are sufficient in extremely small quantities)
- A pair of oligonucleotide primers that have sequences complementary to the ends of the template DNA
- Buffer, Mg2+, etc.
Polymerase chain reaction (PCR) is an in vitro amplification procedure by which DNA fragments of up to 15 kilobases in length can be amplified about 108-fold. In brief, two 10-30 deoxynucleotides long oligonucleotides complementary to nucleotide sequences at the two ends of the target DNA and designed to hybridize opposite strands, are synthesized. The DNA primers are oligonucleotides about 8 to 20 nucleotides long. One primer anneals to the 5′ end of the sense strand, and the other anneals to the 3′ end of the antisense strand of the target sequence. Excessive amounts of these two oligonucleotide primers are mixed with genomic DNA, and the mixture is heated for denaturation of the duplexes.
PCR machine is computerized and so adjusted that at the initial phase, the temperature of the reaction mixture is raised to 94°C when the double-stranded DNA (in the desired gene segment is present) is denatured separating its two strands. The temperature is then cooled down to 50-55°C when the primers will anneal to their genomic homologues in the separated DNA strands and can be extended by Taq DNA polymerase. Following this, the temperature of the reaction mixture is again raised to 70-72°C when Taq polymerase acts over the DNA strand to add dNTPs at the 3′ end of the primer. This sequence of denaturation, annealing of primers, and extension is repeated 20-40 times.
During the second cycle, the target DNA fragment bracketed by the two primers is among the reaction products and serves as a template for subsequent reactions. Thus, repeated cycles of heat denaturation, annealing, and elongation result in an exponential increase in the copy number of the target DNA.
In the thermal cycler, one reaction cycle is usually completed within 2-5 minutes and within less than 3 hours of time 25-30 cycles of reaction may be completed. The use of thermostable DNA polymerases obviates the necessity of adding new polymerases for each cycle. About 25 amplification cycles increase the amount of the target sequence selectively and exponentially by approximately 106-fold.
Advantages of the PCR:
In gene cloning the advantages of PCR are as follows:
- PCR is die most suitable method for cloning of a small segment of DNA located within a quite long DNA molecule.
- With the help of this technique, a large quantity of DNA may be produced from a small quantity of DNA in a very short time. The template DNA required for PCR is very small.
- Cloning of a gene from a degraded DNA is possible in this method.
- From the parts of the decomposed body and even from the fossils of dead organisms, DNA may be obtained for experimental purposes by this technique.
- No RNA primer is required for the polymerization reaction. Oligodeoxyribonucleotides may act as primers in this method.
- The amount of DNA may be produced as per desire.
- Determination of the sequence of genes as well as detection of mutation may be easier as the obtained DNA is large in quantity.
- PCR can amplify specific segments of genes without the need for cloning the segment first.
- No particular expertise of the researcher is required.
Limitations of Polymerase Chain Reaction:
- PCR is much error prone as Taq polymerase has no proofreading property.
- PCR-produced DNA is small in size (15kb), while the size of cell-mediated cloned DNA may reach upto about 2Mb.
- The thermocycler machine and the reagents are expensive.
- PCR does not give satisfactory results if it is not carried out with great care and in controlled conditions.
- Success in PCR depends on prior knowledge of the structure and composition of a gene.
- PCR-produced DNA often contains errors.
Application of PCR:
PCR has many applications such as
- DNA sequencing
- Forensics (PCR is used in forensic medicine to identify victims or criminals by amplifying the minuscule amounts of DNA left at a crime scene).
- Amplification of unknown sequences
- Clinical pathology (PCR can identify infectious diseases such as HIV before symptoms emerge)
- Genetic diagnosis
- Characterizing unknown mutations
- Fingerprinting/population analysis
- Genome analysis
- Molecular cloning
- Quantitative PCR of RNA or DNA.
(ii) Cell-Mediated Gene Cloning
By natural phenomenon when a transformed or transfected cell replicates the introduced recombinant vector containing the desired DNA sequence/gene and produces many copies of the desired gene, this is known as cell-mediated or vivogene cloning. In this regard, an E. coli cell acts as an ideal host cell. However, several other bacterial cells also act as hosts for gene cloning. Among the eukaryotes, Saccharomyces cerevisiae is the widely used host cell. After cloning the genes are isolated by selective techniques as discussed before.