Recombinant DNA and Biotechnology

Brief history of recombinant DNA technology

The present day DNA technology has its roots in the experiments performed by Boyer and Cohen in 1973. In their experiments, they successfully recombined two plasmids (pSC 101 and pSC 102) and cloned the new plasmid in E.coli. In the later experiments the genes of a frog could be successfully transplanted, and expressed in E.coli. This made the real beginning of modern rDNA technology and laid foundations for the present day molecular biotechnology.

Some biotechnologists who admire Boyer-Cohen experiments divide the subject into two chronological categories.

Recombinant DNA technology is a vast field. The basic principles and techniques of rDNA technology along with the most important applications are briefly described in this chapter.

Restriction endonucleases—DNA cutting enzymes

Restriction endonucleases are one of the most important groups of enzymes for the manipulation of DNA. These are the bacterial enzymes that can cut/split DNA (from any source) at specific sites. They were first discovered in E.coli restricting the replication of bacteriophages, by cutting the viral DNA (The host E.coli DNA is protected from cleavage by addition of methyl groups). Thus, the enzymes that restrict the viral replication are known as restriction enzymes or restriction endonucleases.

Cleavage patterns

Majority of restriction endonucleases (particularly type II) cut DNA at defined sites within recognition sequence. A selected list of enzymes, recognition sequences, and their products formed is given in Table 27.1.

Table 27.1

Some restriction enzymes with sources, recognition sequences and the products formed

Enzyme (source)	Recognition sequence	Products
EcoRI		A–A–T–T–C……
(Escherichia coli)		G……
		……G
		C–T–T–A–A
BamHI		G–A–T–C–C……
(Bacillus amyloliquefaciens)		G……
		……G
		……C–C–T–A–G
HaeIII		*C–C……
(Haemophilus aegyptius)		G–G……
		……*G–G
		……C–C
HindIII		A–G–C–T–T……
(Haemophilus influenzae)		A……
		……A
		……T–T–C–G–A
NotI		G–G–C–C–G–C……
(Nocardia otitidis)		C–G……
		……G–C
		……C–G–C–C–G–G……

Note : Scissors indicate the sites of cleavage

^*The products are with blunt ends while for the rest, the products are with sticky ends

The cut DNA fragments by restriction endonucleases may have mostly sticky ends (cohesive ends) or blunt ends, as given in Table 27.1. DNA fragments with sticky ends are particularly useful for recombinant DNA experiments. This is because the single-stranded sticky DNA ends can easily pair with any other DNA fragment having complementary sticky ends.

DNA ligases—DNA joining enzymes

The cut DNA fragments are covalently joined together by DNA ligases. These enzymes were originally isolated from viruses. They also occur in E.coli and eukaryotic cells. DNA ligases actively participate in cellular DNA repair process.

The action of DNA ligases is absolutely required to permanently hold DNA pieces. This is so since the hydrogen bonds formed between the complementary bases (of DNA strands) are not strong enough to hold the strands together. DNA ligase joins (seals) the DNA fragments by forming a phosphodiester bond between the phosphate group of 5’-carbon of one deoxyribose with the hydroxyl group of 3’-carbon of another deoxyribose (Fig.27.2).

Many enzymes are used in the recombinant DNA technology/genetic engineering. A selected list of these enzymes and the reactions catalysed by them is given in Table 27.2.

Prokaryotic hosts

Eukaryotic hosts

Eukaryotic organisms are preferred to produce human proteins since these hosts with complex structure (with distinct organelles) are more suitable to synthesize complex proteins. The most commonly used eukaryotic organism is the yeast, Saccharomyces cerevisiae.

Vectors—the cloning vehicles

Vectors are the DNA molecules, which can carry a foreign DNA fragment to be cloned. They are self-replicating in an appropriate host cell. The most important vectors are plasmids, bacteriophages, cosmids and artificial chromosome vectors.

Plasmid

Plasmids are extrachromosomal, doublestranded, circular, self-replicating DNA molecules. Almost all the bacteria have plasmids containing a low copy number (1–4 per cell) or a high copy number (10–100 per cell). The size of the plasmids varies from 1 to 500 kb. Usually, plasmids contribute to about 0.5 to 5.0% of the total DNA of bacteria (Note : A few bacteria contain linear plasmids e.g. Streptomyces sp, Borella burgdorferi).

pBR322 – the most common plasmid vector

pBR322 of E.coli is the most popular and widely used plasmid vector, and is appropriately regarded as the parent or grand parent of several other vectors.

pBR322 has a DNA sequence of 4,361 bp. It carries genes resistance for ampicillin (Amp^r) and tetracycline (Tet^r) that serve as markers for the identification of clones carrying plasmids. The plasmid has unique recognition sites for the action of restriction endonucleases such as EcoRI, HindIII, BamHI, SalI and PstlI (Fig.27.3).

Bacteriophages

Bacteriophages or simply phages are the viruses that replicate within the bacteria. In case of certain phages, their DNA gets incorporated into the bacterial chromosome and remains there permanently. Phage vectors can accept short fragments of foreign DNA into their genomes. The advantage with phages is that they can take up larger DNA segments than plasmids. Hence phage vectors are preferred for working with genomes of human cells. The most commonly used phages are bacteriophage λ (phage λ) and bacteriophage (phage M₁₃).

Cosmids

Cosmids are the vectors possessing the characteristics of both plasmid and bacteriophage λ. Cosmids can be constructed by adding a fragment of phage λ DNA including cos site, to plasmids. A foreign DNA (about 40 kb) can be inserted into cosmid DNA. The recombinant DNA so formed can be packed as phages and injected into E.coli. Once inside the host cell, cosmids behave just like plasmids and replicate. The advantage with cosmids is that they can carry larger fragments of foreign DNA compared to plasmids.

Artificial chromosome vectors

Choice of vector

Among the several factors, the size of the foreign DNA is very important in the choice of vectors. The efficiency of this process is often crucial for determining the success of cloning. The sizes of DNA insert that can be accepted by different vectors is shown in Table 27.4.

Transformation

Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli). The uptake of plasmid DNA by E.coli is carried out in ice-cold CaCl₂ (0-5°C), and a subsequent heat shock (37–45°C for about 90 sec). By this technique, the transformation frequency, which refers to the fraction of cell population that can be transferred, is reasonably good e.g. approximately one cell per 1000 (10^–3) cells.

Conjugation

Conjugation is a natural microbial recombination process. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges and transfer singlestranded DNA (from donor to recipient). Inside the recipient cell, the new DNA may integrate with the chromosome (rather rare) or may remain free (as is the case with plasmids).

The natural phenomenon of conjugation is exploited for gene transfer. This is achieved by transferring plasmid-insert DNA from one cell to another. In general, the plasmids lack conjugative functions and therefore, they are not as such capable of transferring DNA to the recipient cells. However, some plasmids with conjugative properties can be prepared and used.

Electroporation

Electroporation is based on the principle that high voltage electric pulses can induce cell plasma membranes to fuse. Thus, electroporation is a technique involving electric field-mediated membrane permeabilization. Electric shocks can also induce cellular uptake of exogenous DNA (believed to be via the pores formed by electric pulses) from the suspending solution. Electroporation is a simple and rapid technique for introducing genes into the cells from various organisms (microorganisms, plants and animals).

Liposome-mediated gene transfer

Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic acids. Several techniques have been developed to encapsulate DNA in liposomes. The liposomemediated gene transfer is referred to as lipofection.

On treatment of DNA fragment with liposomes, the DNA pieces get encapsulated inside liposomes. These liposomes can adhere to cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. The positively charged liposomes very efficiently complex with DNA, bind to cells and transfer DNA rapidly.

Direct transfer of DNA

It is possible to directly transfer the DNA into the cell nucleus. Microinjection and particle bombardment are the two techniques commonly used for this purpose.

Lysis of cells

Methods to purify DNA

There are two different approaches to purify DNA from the cellular extracts.

In one method, the addition of a detergent cetyltrimethyl ammonium (CTAB) results in the formation of an insoluble complex with nucleic acids. This complex, in the form of a precipitate is collected after centrifugation and suspended in a high-salt solution to release nucleic acids. By treatment with RNase, RNA is degraded. Pure DNA can be isolated by ethanol precipitation.

The second technique is based on the principle of tight binding between DNA and silica particles in the presence of a denaturing agent such as guanidinium thiocyanate. The isolation of DNA can be achieved by the direct addition of silica particles and guanidinium thiocyanate to the cellular extract, followed by centrifugation. Alternately, a column chromatography containing silica can be used, and through this the extract and guanidinium thiocyanate are passed. The DNA binds to the silica particles in the column which can be recovered.

Types of blotting techniques

The most comonly used blotting techniques are listed below

The Southern blotting is named after the scientist Ed Southern (1975) who developed it. The other names Northern blotting and Western blotting are laboratory jargons which are now accepted. Western blotting involves the transfer of protein blots and their identification by using specific antibodies.

A diagrammatic representation of a typical blotting apparatus is depicted in Fig.27.7.

Applications of Southern blotting

Western blotting

Western blotting involves the identification of proteins. It is very useful to understand the nucleic acid functions, particularly during the course of gene manipulations.

The technique of Western blotting involves the transfer of electrophoresed protein bands from polyacrylamide gel to nylon or nitrocellulose membrane. These proteins can be detected by specific protein-ligand interactions. Antibodies or lectins are commonly used for this purpose.

Autoradiography

Autoradiography is the process of localization and recording of a radiolabel within a solid specimen, with the production of an image in a photographic emulsion. These emulsions are composed of silver halide crystals suspended in gelatin.

When a β-particle or a γ-ray from a radiolabel passes through the emulsions, silver ions are converted to metallic silver atoms. This results in the development of a visible image which can be easily detected.

Applications of autoradiography

As already described, autoradiography is closely associated with blotting techniques for the detection of DNA, RNA and proteins.

Technique of DNA sequencing

A DNA chip carrying an array of different oligonucleotides can be used for DNA sequencing. For this purpose, a fluorescently labeled DNA test molecule, whose sequence is to be determined, is applied to the chip. Hybridization occurs between the complementary sequences of the test DNA molecule and oligonucleotides of the chip. The positions of these hybridizing oligonucleotides can be determined by confocal microscopy. Each hybridizing oligonucleotide represents an 8- nucleotide sequence that is present in the DNA probe. The sequence of the test DNA molecule can be deduced from the overlaps between the sequences of the hybridizing oligonucleotides (Fig.27.14).

Applications of DNA chips

There have been many successes with this relatively new technology of DNA chips. Some of them are listed.

The future of DNA chips

The major limitation of DNA chips at present is the unavailability of complete genome arrays for higher eukaryotes, including humans. It is expected that within the next few years such DNA chips will be available. This will help the biotechnologists to capture the functional snapshots of the genome in action for higher organisms.

Principle of PCR

The double-stranded DNA of interest is denatured to separate into two individual strands. Each strand is then allowed to hybridize with a primer (renaturation). The primer-template duplex is used for DNA synthesis (the enzyme-DNA polymerase). These three steps— denaturation, renaturation and synthesis are repeated again and again to generate multiple forms of target DNA.

Technique of PCR

The essential requirements for PCR are listed below

The actual technique of PCR involves repeated cycles for amplification of target DNA. Each cycle has three stages.

The 3 stages of PCR in relation to temperature and time are depicted in Fig.27.15. Each cycle of PCR takes about 3–5 minutes. In the normal practice, the PCR is carried out in an automated machine.

As is evident from the Fig.27.16 (cycle I), the new DNA strand joined to each primer is beyond the sequence that is complementary to the second primer. These new strands are referred to as long templates, and they will be used in the second cycle.

For the second cycle of PCR, the DNA strands (original + newly synthesized long template) are denatured, annealed with primers and subjected to DNA synthesis. At the end of second round, long templates, and short templates (DNA strands with primer sequence at one end, and sequence complementary to the other end primer) are formed.

In the third cycle of PCR, the original DNA strands along with long and short templates are the starting materials. The technique of denaturation, renaturation and synthesis are repeated. This procedure is repeated again and again for each cycle. It is estimated that at the end of 32^nd cycle of PCR, about a million-fold target DNA is synthesized. The short templates possessing precisely the target DNA as doublestranded molecules accumulate.

Sources of DNA polymerase

In the original technique of PCR, Klenow fragment of E. coli DNA polymerase was used. This enzyme, gets denatured at higher temperature, therefore, fresh enzyme had to be added for each cycle. A breakthrough occurred (Lawyer 1989) with the introduction of Taq DNA polymerase from thermophilic bacterium, Thermus aquaticus. The Taq DNA polymerase is heat resistant, hence it is not necessary to freshly add this enzyme for each cycle of PCR.

Variations of PCR

The basic technique of PCR has been described. Being a versatile technique, PCR is modified as per the specific demands of the situation. Some of the variants of PCR are listed

PCR in clinical diagnosis

The specificity and sensitivity of PCR is highly useful for the diagnosis of various diseases in humans. These include diagnosis of inherited disorders (genetic diseases), viral diseases, bacterial diseases etc.

PCR in DNA sequencing

As the PCR technique is much simpler and quicker to amplify the DNA, it is conveniently used for sequencing. For this purpose, singlestrands of DNA are required.

PCR in comparative studies of genomes

The differences in the genomes of two organisms can be measured by PCR with random primers. The products are separated by electrophoresis for comparative identification. Two genomes from closely related organisms are expected to yield more similar bands.

PCR is very important in the study of evolutionary biology, more specifically referred to as phylogenetics. As a technique which can amplify even minute quantities of DNA from any source (hair, mummified tissues, bone, or any fossilized material), PCR has revolutionized the studies in palaentology and archaelogy. The movie ‘Jurassic Park’, has created public awareness of the potential applications of PCR!

PCR in forensic medicine

A single molecule of DNA from any source (blood strains, hair, semen etc.) of an individual is adequate for amplification by PCR. Thus, PCR is very important for identification of criminals.

The reader may refer DNA finger printing technique described later in this chapter.

Establishing a gene library for humans

The human cellular DNA (the entire genome) may be subjected to digestion by restriction endonucleases (e.g., EcoRI). The fragments formed on an average are of about 4 kb size. (i.e., 4000 nitrogenes bases). Each human chromosome, containing approximately 100,000 kb can be cut into about 25,000 DNA fragments. As the humans have 23 different chromosomes (24 in man), there are a total of 575,000 fragments of 4 kb length formed. Among these 575,000 DNA fragments is the DNA or gene of interest (say insulin gene).

Now is the selection of a vector and cloning process. E.coli, a harmless bacterium to humans is most commonly used. The plasmids from E. coli are isolated. They are digested by the same restriction enzyme as was used for cutting human genome to form open plasmids. The human chromosomal DNA fragments and open plasmids are joined to produce recombined plasmids. These plasmids contain different DNA fragments of humans. The recombined plasmids are inserted into E. coli and the cells multiply (Fig.27.17). The E. coli cells possess all the human DNA in fragments. It must, however be remembered that each E. coli cell contains different DNA fragments. All the E. coli cells put together collectively represent genomic library (containing about 575,000 DNA fragments).

Screening strategies

Once a DNA library is created, the clones (i.e., the cell lines) must be screened for identification of specific clones. The screening techniques are mostly based on the sequence of the clone or the structure/function of its product.

Screening by DNA hybridization

The target sequence in a DNA can be determined with a DNA probe (Fig.27.18). To start with, the double-stranded DNA of interest is converted into single strands by heat or alkali (denaturation). The two DNA strands are kept apart by binding to solid matrix such as nitrocellulose or nylon membrane. Now, the single strands of DNA probe (100–1,000 bp) labeled with radioisotope are added. Hybridization (i.e., base pairing) occurs between the complementary nucleotide sequences of the target DNA and the probe. For a stable base pairing, at least 80% of the bases in the two strands (target DNA and the probe) should be matching. The hybridized DNA can be detected by autoradiography.

(Note : DNA probe or gene probe represents a segment of DNA that is tagged with a label (i.e. isotope) so as to detect a complementary base sequence with sample DNA after hybridization)

Tissue plasminogen activator (tPA)

Tissue plasminogen activator is therapeutically used to lyse the blood clots that cause myocardial infarction. Due to its shorter half-life (around 5 minutes), tPA has to be repeatedly administered. By replacing asparagine residue (at position 120) with glutamine, the half-life of tPA can be substantially increased. This is due to the fact that glutamine is less glycosylated than asparagine and this makes a difference in the half-life of tPA.

Hirudin

Hirudin is a protein secreted by leech salivary gland, and is a strong thrombin inhibitor (i.e., acts as an anticoagulant). By replacing asparagine (at 47 position) with lysine, the potency of hirudin can be increased severalfold.

Nucleic acid hybridization

Hybridization of nucleic acids (particularly DNA) is the basis for reliable DNA analysis. Hybridization is based on the principle that a single-stranded DNA molecule recognizes and specifically binds to a complementary DNA strand amid a mixture of other DNA strands. This is comparable to a specific key and lock relationship. The general procedure adopted for nucleic acid hybridization has been described (See p. 597 and Fig.27.18). Some more information is given below (Fig.27.19).

The single-stranded target DNA is bound to a membrane support. Now the DNA probe (single-stranded and labeled with a detector substance) is added. Under appropriate conditions (temperature, ionic strength), the DNA probe pairs with the complementary target DNA. The unbound DNA probe is removed. Sequence of nucleotides in the target DNA can be identified from the known sequence of DNA probe.

There are two types of DNA hybridizationradioactive and non-radioactive respectively using DNA probes labeled with isotopes and non-isotopes as detectors.

Technique for use of DNA chip

The unknown DNA molecules are cut into fragments by restriction endonucleases. Fluorescent markers are attached to these DNA fragments. They are allowed to react with the probes of the DNA chip. Target DNA fragments with complementary sequences bind to DNA probes. The remaining DNA fragments are washed away. The target DNA pieces can be identified by their fluorescence emission by passing a laser beam. A computer is used to record the pattern of fluoresence emission and DNA identification.

The technique of employing DNA chips is very rapid, besides being sensitive and specific for the identification of several DNA fragments simultaneously. Scientists are trying to develop Genechips for the entire genome of an organism.

Applications of DNA chip

The presence of mutations in a DNA sequence can be conveniently identified. In fact, Genechip probe array has been successfully used for the detection of mutations in the p53 and BRCA I genes. Both these genes are involved in cancer (See p. 593 also).

Tuberculosis

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis. The commonly used diagnostic tests for this disease are very slow, and sometimes may take several weeks. This is because M. tuberculosis multiplies very slowly (takes about 24 hrs. to double; E. coli takes just 20 minutes to double).

A novel diagnostic test for tuberculosis was developed by genetic engineering, and is illustrated in Fig.27.20. A gene from firefly, encoding the enzyme luciferase is introduced into the bacteriophage specific for M. tuberculosis. The bacteriophage is a bacterial virus, frequently referred to as luciferase reporter phage or mycophage. The genetically engineered phage is added to the culture of M. tuberculosis. The phage attaches to the bacterial cell wall, penetrates inside, and inserts its gene (along with luciferase gene) into the M. tuberculosis chromosome. The enzyme luciferase is produced by the bacterium. When luciferin and ATP are added to the culture medium, luciferase cleaves luciferin. This reaction is accompanied by a flash of light which can be detected by a luminometer. This diagnostic test is quite sensitive for the confirmation of tuberculosis.

The flash of light is specific for the identification of M. tuberculosis in the culture. For other bacteria, the genetically engineered phage cannot attach and enter in, hence no flash of light would be detected.

Malaria

Malaria, mainly caused by Plasmodium falciparum, and P. vivax, affects about one-third of the world's population. The commonly used laboratory tests for the diagnosis of malaria include microscopic examination of blood smears, and detection of antibodies in the circulation. While the former is time consuming and frequently gives false-negative tests, the latter cannot distinguish between the past and present infections.

A specific DNA diagnostic test for identification of the current infection of P. falciparum has been developed. This is carried out by using a DNA probe that can bind and hybridize with a DNA fragment of P. falciparum genome, and not with other species of Plasmodium. It is reported that this DNA probe can detect as little as 1 ng of P. falciparum in blood or 10 pg of its purified DNA.

Acquired immunodeficiency syndrome (AIDS)

DNA probes, with radioisotope label, for HIV DNA are now available. By using PCR and DNA probes, AIDS can be specifically diagnosed in the laboratory.

Sickle-cell anemia

Sickle-cell anemia is a genetic disease characterized by the irregular sickle (crescent like) shape of the erythrocytes. Biochemically, this disease results in severe anemia and progressive damage to major organs in the body (heart, brain, lungs, joints).

Sickle-cell anemia occurs due to a single amino acid change in the β-chain of hemoglobin. Specifically, the amino acid glutamate at the 6th position of β-chain is replaced by valine. At the molecular level, sickle-cell anemia is due to a single-nucleotide change (A → T) in the β-globin gene of coding (or antisense) strand. In the normal β-globin gene the DNA sequence is CCTGAGGAG, while in sickle-cell anemia, the sequence is CCTGTGGAG. This single-base mutation can be detected by using restriction enzyme MstII to cut DNA fragments in and around β-globin gene, followed by the electrophoretic pattern of the DNA fragments formed. The change in the base from A to T in the β-globin gene destroys the recognition site (CCTGAGG) for MstII (Fig.27.21). Consequently, the DNA fragments formed from a sickle-cell anemia patient for β-globin gene differ from that of a normal person. Thus, sickle-cell anemia can be detected by digesting mutant and normal β-globin genes by restriction enzyme and performing a hybridization with a cloned β-globin DNA probe.

History and terminology

The original DNA fingerprinting technique was developed by Alec Jaffreys in 1985. Although the DNA fingerprinting is commonly used, a more general term DNA profiling is preferred. This is due to the fact that a wide range of tests can be carried out by DNA sequencing with improved technology.

Applications of DNA fingerprinting

The amount of DNA required for DNA fingerprint is remarkably small. The minute quantities of DNA from blood strains, body fluids, hair fiber or skin fragments are enough. Polymerase chain reaction is used to amplify this DNA for use in fingerprinting. DNA profiling has wide range of applications—most of them related to medical forensics. Some important ones are listed below.

In general, the fingerprinting technique is carried out by collecting the DNA from a suspect (or a person in a paternity or immigration dispute) and matching it with that of a reference sample (from the victim of a crime, or a close relative in a civil case).

RFLPs in the diagnosis of diseases

If the RFLP lies within or even close to the locus of a gene that causes a particular disease, it is possible to trace the defective gene by the analysis of RFLP in DNA. The person's cellular DNA is isolated and treated with restriction enzymes. The DNA fragments so obtained are separated by electrophoresis. The RFLP patterns of the disease suspected individuals can be compared with that of normal people (preferably with the relatives in the same family). By this approach, it is possible to determine whether the individual has the marker RFLP and the disease gene. With 95% certainity, RFLPs can detect single gene-based diseases.

Methods of RFLP scoring

Two methods are in common use for the detection of RFLPs (Fig.27.23).

1. Southern hybridization : The DNA is digested with appropriate restriction enzyme, and separated by agarose gel electrophoresis. The so obtained DNA fragments are transferred to a nylon membrane. A DNA probe that spans the suspected restriction site is now added, and the hybridized bands are detected by autoradiograph. If the restriction site is absent, then only a single restriction fragment is detected. If the site is present, then two fragments are detected (Fig.27.23A).

2. Polymerase chain reaction : RFLPs can also be scored by PCR. For this purpose, PCR primers that can anneal on either side of the suspected restriction site are used. After amplification by PCR, the DNA molecules are treated with restriction enzyme and then analysed by agarose gel electrophoresis. If the restriction site is absent only one band is seen, while two bands are found if the site is found (Fig.27.23B).

Use of RFLPs and VNTRs in genetic fingerprinting

RFLPs caused by variations in the number of VNTRs between two restriction sites can be detected (Fig.27.25). The DNAs from three individuals with different VNTRs are cut by the specific restriction endonuclease. The DNA fragments are separated by electrophoresis, and identified after hybridization with a probe complementary to a specific sequence on the fragments.

Production of recombinant insulin

Attemps to produce insulin by recombinant DNA technology started in late 1970s. The basic technique consisted of inserting human insulin gene and the promoter gene of lac operon on to the plasmids of E. coli. By this method human insulin was produced. It was in July 1980, seventeen human volunteers were, for the first time, administered recombinant insulin for treatment of diabetes at Guy's Hospital, London. And in fact, insulin was the first ever pharmaceutical product of recombinant DNA technology administered to humans. Recombinant insulin worked well, and this gave hope to scientists that DNA technology could be successfully employed to produce substances of medical and commercial importance. An approval, by the concerned authorities, for using recombinant insulin for the treatment of diabetes mellitus was given in 1982. And in 1986, Eli Lilly company received approval to market human insulin under the trade name Humulin.

Technique for production of recombinant insulin

The orginal technique (described briefly above) of insulin synthesis in E. coli has undergone several changes, for improving the yield. e.g. addition of signal peptide, synthesis of A and B chains separately etc.

The procedure employed for the synthesis of two insulin chains A and B is illustrated in Fig.27.28. The genes for insulin A chain and B chain are separately inserted to the plasmids of two different E. coli cultures. The lac operon system (consisting of inducer gene, promoter gene, operator gene and structural gene Z for β-galactosidase) is used to express both the genes. The presence of lactose in the culture medium induces the synthesis of insulin A and B chains in separate cultures. The so formed insulin chains can be isolated, purified and joined together to give a full-fledged human insulin.

Hepatitis B vaccine—the first synthetic vaccine

In 1987, the recombinant vaccine for hepatitis B (i.e. HBsAg) became the first synthetic vaccine for public use. It was marketed by trade names Recombivax and Engerix-B. Hepatitis B vaccine is safe to use, very effective and produces no allergic reactions. For these reasons, this recombinant vaccine has been in use since 1987. The individuals must be administered three doses over a period of six months. Immunization against hepatitis B is strongly recommended to anyone coming in contact with blood or body secretions. All the health professionals—physicians, surgeons, medical laboratory technicians, nurses, dentists, besides police officers, firefighters etc., must get vaccinated against hepatitis B.

Hepatitis B vaccine in India

India is the fourth country (after USA, France and Belgium) in the world to develop an indigenous hepatitis B vaccine. It was launched in 1997, and is now being used.

Humoral immunity

As the antigens bind to B-lymphocytes, they trigger the production of antibodies which can destroy the pathogens. Some of the B-lymphocytes become memory cells that can protect the host against future infections.

Cellular immunity

The protein fragments of the antigen bound to MHC molecules can activate the cytotoxic T-lymphocytes. They are capable of destroying the infected pathogenic cells. Some of the activated T-lymphocytes become memory cells which can kill the future infecting pathogens.

Importance of transgenic animals—general

Transgenesis has now become a powerful tool for studying the gene expression and developmental processes in higher organisms, besides the improvement in their genetic characteristics. Transgenic animals serve as good models for understanding the human diseases. Further, several proteins produced by transgenic animals are important for medical and pharmaceutical applications. Thus, the transgenic farm animals are a part of the lucrative world-wide biotechnology industry, with great benefits to mankind.

Risks and ethics of biotechnology

The modern biotechnology deals with genetic manipulations of viruses, bacteria, plants, animals, fish and birds. Introduction of foreign genes into various organisms raises concerns about the safety, ethics and unforeseen consequences. Some of the popular phrases used in the media while referring to experiments on recombinant DNA technology are listed.

The major apprehension of genetic engineering is that through recombinant DNA experiments, unique microorganisms or viruses (either inadvertently, or sometimes deliberately for the purpose of war) may be developed that would cause epidemics and environmental catastrophes. Due to these fears, the regulatory guidelines for research dealing with DNA manipulation were very stringent in the earlier years.

So far, risk assessment studies have failed to demonstrate any hazardous properties acquired by host cells/organisms due to transfer of DNA. Thus, the fears of genetic manipulations may be unfounded to a large extent. Consequently, there has been some relaxation in the regulatory guidelines for recombinant DNA research.

It is now widely accepted that biotechnology is certainly beneficial to humans. But it should not cause problems of safety to people and environment, and create unacceptable social, moral and ethical issues.