Introduction to genetic engineering

About genes
Genes are at the very heart of life. Together they constitute the blueprint of an organism. In computer terms they are
the master program of life. They decide all the properties and all the capabilities of an organism.
In biological terms this master program is called the hereditary substance, the chromosomes. It is constituted by
chains of so called DNA molecules that carry the "code words" or instructions of the master program.
There is an identical set of this master program in every cell. For example a corn plant has about a billion cells, each
with a set of this master program. In different parts of the plants different parts of the program are active, giving rise to
different structures like the leaves, the seeds and the root. The cell is like a huge computer network, much larger than
any man-made one. Science has a very incomplete understanding how this billion of master programs is able to
cooperate in a very harmoniously and effectively coordinated way.
Genetic engineering
Genetic engineering (also called gene modification) means manipulation of this master program. Genes, mostly from
other, often totally unrelated species are inserted in the genetic "master program". Genes from e.g. fish, scorpions,
bacteria and viruses have been inserted into food plants in genetic engineering projects.
Genetic engineering is based on an outdated theory proven to be wrong. It stated that one gene carries one
property. This is not so. The effect of a gene is dependent on its location and its interaction with other genes.
Therefore, insertion of foreign genes is bound to cause unpredictable surprises including, in the worst case,
the appearance of harmful substances in the food.
Moreover, the method of genetic engineering is so crude that it is impossible to decide beforehand where the inserted
genes will stick in the master program. This adds further to the unpredictability of the outcome of artifical gene
insertion (genetic engineering).
In spite of this inevitable unpredictability, the safety of GE foods has not been tested in such a way that
unexpected harmful substances are ruled out.
The knowledge about genes is very incomplete
In addition, the knowledge about the master program is very incomplete. Actually only 2-3 percent of it are so called
genes. Their function is fairly well known. The function and purpose of the remaining 97-98 percent is very little
known.
From genetics it is well known that even changing just a little code word in the master program can mean the
difference between health and a deadly hereditary disease. So the genes are very powerful.
It is probably not a coincidence that an unproportionately large part of our members are computer experts. They know
that the addition of just one "code syllable" (binary code) may be disastrous to a computer program. Haphazard
insertion of genes, as done in genetic engineering, does not add just one syllable, but many thousand of code
syllables. In addition, it is obvious to a computer scientist that it is absolutely vital to completely master the program in
order to be able to make a useful change in a reliable way.
History of GE:
The history of genetic engineering can be traced back to the prehistoric times when man used selective breeding and
cross breeding to develop better species of food grains and livestock. The mule is one example. It is a cross between
a male donkey and a female horse, which was developed by the process of interspecies breeding and has been in
existence for thousands of years now. Darwin's famous book, The Origin of Species tells us a lot about what all the
people of the time knew about breeding. Today, genetic engineering is advancing at such a rate that there will soon
come a time when nothing would be impossible for man. Let's have a look at the genetic engineering history timeline
to know about the milestones achieved in this field.
History of Genetic Engineering in Animals
Here we shall have a look at the major events in the field of genetic engineering that contributed to the development
of new species of animals as well as advancements in the field of medicine. We would begin with the 19th century
and would gradually proceed to the present.
1859 The Origin of Species by Charles Darwin was published and it gave an account of the knowledge people had
about selective breeding.
1865 Gregor Mendel laid the foundation of modern genetics with his pathbreaking experiment of crossbreeding pea
plants (Pisum sativum) with different characteristics and his observations served as the basis for the principles of
genetics.
1866 Ernst Haeckel discovered that the genetic material of a cell resides in its nucleus.
1890 First animal, a rabbit, was created by the process of in-vitro fertilization (IVF).

1991 Gene therapy was first tried and tested on humans. 1902 The chromosome theory of inheritance was proposed by Walter Sutton & Theodor Boveri. Artificial insemination was carried out for the first time in humans. Celera Genomics. 1977 Sequence of bases in DNA was discovered by Walter Gilbert and Frederick Sanger. 1973 The first experiment on recombinant DNA cloning was performed by Herbert Boyer and Stanley Cohen. 1931 The phenomenon of physical recombination of DNA was discovered. 1968 Discovery of endonucleases or DNA "cutting" enzymes was done by Stewart Linn and Werner Arber. 1966 Unraveling of the genetic code was done by Marshall Nirenberg and Har Gobind Khorana. 1944 By carrying out experiments on bacteria. 1958 Semiconservative nature of DNA replication was established. was a sheep born from a mammary cell of an adult sheep as nucleus donor and an enucleated ovum as recipient. 2000 On the 26th June. was discovered. 1990 Launching of the Human Genome Project to map the entire human genome. the leaders of the publicly sponsored Human Genome Project (HGP) and the company. It was a landmark achievement in the field of genetic engineering in humans. 1910 T. Oswald Avery established the role of DNA in genetics. 1953 James Watson and Francis Crick proposed the double helix structure of DNA for the first time. was done by James Watson and others. H. Erich von Tschermak and Carl Correns. 1978 through in vitro fertilization (IVF). 1984 Birth of a human baby took place from frozen embryo. was transplanted into baboons. announced the completion of the first draft of the human genome. 1986 Embryo cells from sheep were cloned. 2000. While studying the symptoms of a disease known as alkaptonuria. 1941 The role of enzymes in the growth of an organism was established by George Beadle and E. Tatum. Archibald Garrod learned that defects in enzymes and enzyme secretion are caused by defective genes. 1976 Prenatal genetic diagnosis with the help of DNA. L. 1979 Method of producing insulin using genetic engineering. 1983 Polymerase chain reaction in DNA was discovered by Kary Mullis. Morgan proved that genetic material is present within the chromosome. Louise Brown was born on 25th July. However. was discovered. he was banned from going ahead with the experiment. 1980 The first genetically modified mouse was developed. 1987 Transgenic mice were developed that were born with human genes. the first cloned animal. 1998 Lee Bo-yon of Kyunghee University in South Korea claimed to have successfully developed the first human clone. 1978 The world's first test tube baby. 1997 Dolly. .1900 Mendel's principles of genetics were rediscovered by Hugo de Vries. 1995 Heart of a genetically modified pig that contained human genes.

let's have a look at the important events in the history of genetic engineering in agriculture. erythropoietin. 2004 First 'true' human clones were developed at the Seoul National University in South Korea. creates condition for the insulin to express itself to produce insulin through the normal process of transfer of information from DNA to protein. Then ensures that these capabilities are converted into abilities. Genetically engineering plants are also poised to produce vaccines. to improve the characteristics of plant species. the genetic material) of another having these capabilities does this. Genetic engineering implies conferring new capabilities on an organism by Transferring into an organism the appropriate DNA (De oxyribo Nucleic Acid. 1994 Transgenic tomatoes were released in the market for the first time. They will be available at a cost of three or more times lower than the current cost. Food and Drug Administration (FDA) approved the first genetically modified food. 1973 Ti plasmid. Transgenic organisms are created using recombinant DNA. 1952 First in-vitro or test-tube plants were developed. human insulin. Now. The total acreage under genetically engineered crops (for good or for bad) around the world exceeds 100 million acres today. but we can make it to do so by introducing in it the gene for human insulin (that is. Roundup.are already in the market. the appropriate DNA fragment coding for this protein). 1900 Using Gregor Mendel's principles of genetics. 1987 The first field tests of genetically engineered crops (tobacco and tomato) were conducted in the United States. For example genetically engineered plants that make their own pesticides or are resistant to weedicides.2001 Birth of the world's first genetically modified human babies. Knowledge of GE: What is genetic engineering? A method of cutting DNA from one organism and inserting the DNA fragments into a host organism of the same or different species. USA. . What is a Transgenic Organism? Transgenic Organisms – organisms that contain foreign DNA (DNA from another organism).S. Sacchromyces cerevisciae cannot make the protein. History of Genetic Engineering in Agriculture. scientists in Europe developed a process termed as "classic selection". would be genetically engineered animals who would secrete these drugs in abundance (1-15 mg/ml) in their milk. One of the future sources of cheap protein-drugs in the coming years. plants and animals (including marine animals). The first ever human clone was successfully developed at the Advanced Cell Technologies. and hepatitis-B vaccine). Thus. was first developed. Scope and basic knowledge of Genetic Engineering: Genetic Engineering of microbes. Thus the common yeast. After integrating the insulin gene in yeast DNA. 1988 The first transgenic corn was developed. 1983 First transgenic plant (tobacco) was developed. 1990 U. Genetically engineered microbes are today widely used for producing drugs and vaccines in large scale at low costs that are of great importance (human insulin. took place. Recombinant DNA = DNA made by connecting or recombining fragments of DNA from different sources. which is used for genetically engineering plants. which was a type of cross breeding. A few hundred acres of genetically engineered banana plantation can provide enough vaccine to immunize 120 million children every year that need to be protected against four common diseases. over 60 percent of the acreage under soyabean in the United States have now genetically engineered soyabean that is resistant to the weedicides.

(iii) selection of hybrid cells. elementary consideration of these steps is presented below. Step three of genetic engineering involves the recombinant DNA plasmid being inserted back into the bacterial cell (mixed with millions of bacteria suspended in a dense salt solution). DNA fragments cannot function all by themselves. They are the first patented organisms. the bacteria will take up the recombinant DNA. Protoplast Fusion: The techniques for protoplast fusion are pretty well refined and highly effective for almost all the systems. The cuts made by the restriction enzymes produce the same “sticky ends” on the DNA and the cut plasmids. The term for a large number of cells grown from a single cell is a clone. Scientists have modified the bacterium E. . Automobile fuel from discarded corn stalks. They must become part of the genetic material of living cells before the genes they contain can be activated. and such hybrids are known as somatic hybrids. Create bacteria that can break down oil from oil spills faster than normal bacteria. A number of strategies have been used to induce fusion between protoplasts of different strains/species. (ii) fusion of the protoplasts of desired species/varieties. and (iv) culture of the hybrid cells and regeneration of hybrid plants from them. DNA fragments may be combined with bacterial DNA so they can later be inserted into a bacterial cell. and sewage treatment. These bacteria can be isolated and grown into large colonies that contain recombinant DNA. Bacteria contain small circular DNA molecules known as plasmids in addition to their chromosomes. A brief. pulp and paper products. The technique of somatic hybridization involves the following four steps: (i) isolation of protoplasts. therefore this technique is often referred to as DNA cloning. of these the following three (Fig. After a few minutes. These plasmids can be removed from the bacterial cells and cut with the same restriction enzyme used to produce the DNA fragments. These sticky ends are the sites at which the DNA fragment and the plasmid can be joined end to end (paste). PROTOPLAST FUSION AND SOMATIC HYBRIDIZATION Somatic Hybridization of Hybrid Plants (explained with diagram)! Production of hybrid plants through the fusion of protoplasts of two different plant species/varieties is called somatic hybridization. Production of cheese.10) have been relatively more successful. 8. thereby forming a new plasmid that contains a piece of foreign DNA (recombinant DNA). In the second step of genetic engineering the DNA fragments are incorporated into part of the recipient cell’s genetic material. laundry detergent. coli to produce expensive indigo dye that is used to color blue genes.How is recombinant DNA made? Cut and Paste (Just like word processing) Restriction enzymes (bacterial proteins) cut DNA (like scissors) at specific sequences (the gene you want) from the original organism’s chromosome. High protein corn with protein levels similar to beef.

it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. in turn. and this population is called a DNA clone. This technique is quite suitable for some species. PEG is negatively charged and may bind to cation like Ca2+. The washing medium may be alkaline (pH 9-10) and contain a high Ca2+ ion concentration (50 m mol l-1). During the washing process. The protoplast mixture is treated with 28-50% PEG (MW 1. A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host.5) and high Ca2+ concentration (50 m mol l-1) at 37°C for about 30 min (high pH-high Ca2+ treatment). The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus). or vectors.Protoplasts of desired strains/species are mixed in almost equal proportion. Later. they can also bind to cationic molecules of plasma membrane. all carrying the same recombinant vector. for the DNA fragments. and it is common for single recombinant vector molecules to find their way into individual bacterial cells. may bind to the negatively charged molecules present in plasma lemma. Recombinant DNA technology How does recombinant DNA technology work? The organism under study. which. These small circular molecules act as carriers. followed by gradual washing of the protoplasts to remove PEG. however. which will be used to donate DNA for the analysis. while for some others it may be toxic. The recombinant DNA mixture is then used to transform bacterial cells. they are mixed while still suspended in the enzyme mixture. generally. this approach is a combination of PEG and high pH-high Ca2+ treatments. protoplast fusion occurs during the washing. Therefore an individual colony contains a very large population of identical DNA inserts. PEG molecules may pull out the plasma lemma components bound to them. The protoplast mixture is then subjected to high pH (10. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells. Bacterial cells are plated and allowed to grow into colonies. This would disturb plasma lemma organisation and may lead to the fusion of protoplasts located close to each other. is called the donor organism.000) for 15-30 min. Hence the protocol is often as follows: .500-6. and is usually more effective than either treatment alone. \ Polyethylene glycol (PEG) induced protoplast fusion is the most commonly used as it induces reproducible high frequency fusion accompanied with low toxicity to most cell types.

Donor DNA from any other source (say. Restriction sites are not relevant to the function of the organism. at a specific alkaline pH. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. and the linear molecule formed has two sticky ends. Production of these sticky ends is another feature of restriction enzymes that makes them suitable for recombinant DNA technology. both will produce fragments with the same complementary sticky ends. and they would not be cut in vivo. making it possible for DNA chimeras to form. contains restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments of a size suitable for cloning. Let’s look at an example: the restriction enzyme EcoRI (from E. This staggered cut leaves a pair of identical single-stranded “sticky ends. cutting up the DNA of the phage and thereby inactivating it. the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes. The procedure used for obtaining vector DNA depends on the nature of the vector. The principle is simply that. bacterial genomic DNA denatures but plasmids do not. The enzyme EcoRI cuts within this sequence but in a pair of staggered cuts between the G and the A nucleotides. rather.Isolation of DNA: The first step in making recombinant DNA is to isolate donor and vector DNA. Any DNA molecule. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate. both donor DNA and vector DNA are digested with the use of a restriction enzyme that produces sticky ends and then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind to each other and form recombinant molecules. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology. coli) recognizes the following six-nucleotidepair sequence in the DNA of any organism: This type of segment is called a DNA palindrome. from viral to human. The plasmid band is collected by punching a hole in the plastic centrifuge tube. The enzymes act like scissors. if both vector DNA and donor DNA are cut with EcoRI. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 12-2.” The ends are called sticky because they can hydrogen bond (stick) to a complementary sequence. Restriction enzymes are produced by bacteria as a defense mechanism against phages. so digestion with the restriction enzyme EcoRI converts the circular DNA into a linear molecule with sticky ends. Bacterial plasmids are commonly used vectors. Importantly. they cut at specific DNA target sequences. Subsequent neutralization precipitates the genomic DNA. EcoRI making a single cut in a circular DNA molecule such as a plasmid: the cut opens up the circle. if two different DNA molecules are cut with the same restriction enzyme. because most organisms do not have restriction enzymes. Drosophila) also is treated with the EcoRI enzyme to produce a population of fragments carrying . but plasmids stay in solution. which means that both strands have the same nucleotide sequence but in antiparallel orientation. Cutting DNA The breakthrough that made recombinant DNA technology possible was the discovery and characterization of restriction enzymes. With the use of such methods. Many different restriction enzymes recognize and cut specific palindromes. Hence. which is one of the key features that make them suitable for DNA manipulation. Plasmid vector that carries a single EcoRI restriction site. these types are generally the ones required for analysis. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. Another protocol relies on the observation that. the sticky ends of the vector can bond to the sticky ends of a donor fragment when the two are mixed. Most commonly. and these plasmids must be purified away from the bacterial genomic DNA. restriction enzymes do not cut randomly.

However.the same sticky ends. the sugar-phosphate backbones are still not complete at two positions at each junction. the backbones can be sealed by the addition of the enzyme DNA ligase. because double helices form between their sticky ends. At this stage. There are many opened-up vector molecules in the solution. and many different EcoRI fragments of donor DNA. When the two populations are mixed. DNA fragments from the two sources can unite. Therefore a diverse array of vectors carrying different donor inserts will be produced. which create phosphodiester bonds at the junctions. although sticky ends have united to generate a population of chimeric molecules. . Certain ligases are even capable of joining DNA fragments with blunt-cut ends.