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Chapter Contents Aakash Educational Services Limited - Regd. Office : Aakash Tower, 8, Pusa Road, New Delhi-110005 Ph. 011-47623456 In its purest form, the term biotechnology refers to the use of living organisms or their products to modify human health and the human environment. However, it is used in a restricted sense today, to refer to those processes which use genetically modified organisms to achieve the same goal but on a larger scale. Many other processes/techniques are also included under biotechnology. For example, in-vitro fertilisation leading to a ‘test-tube’ baby, synthesising a gene and using it, developing a DNA vaccine or correcting a defective gene, are all part of biotechnology. The European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional views and modern molecular biotechnology. The definition given by EFB is as follows : The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services. PRINCIPLES OF BIOTECHNOLOGY The two core techniques that enabled us to combine the genetic elements of two or more living cells or that enabled birth of modern biotechnology are : (i) Genetic Engineering : Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. (ii) Bioprocess engineering : Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes etc. In these techniques functioning lengths of DNA can be taken from one organism and placed into the cells of another organism. As a result, we can cause bacterial cells to produce human molecules. Cows can produce more milk for the same amount of feed. And we can synthesize therapeutic molecules that have never existed before. Principles of Biotechnology Tools of Recombinant DNA Technology Processes of Recombinant DNA Technologies Chapter 18 Biotechnology - Principles and Processes
72 Biotechnology - Principles and Processes NEET Aakash Educational Services Limited - Regd. Office : Aakash Tower, 8, Pusa Road, New Delhi-110005 Ph. 011-47623456 Let us now understand the conceptual development of the principles of genetic engineering. Traditional hybridisation procedures used in plant and animal breeding, very often lead to inclusion and multiplication of undesirable genes along with desired genes. The techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer overcome this limitation and allow us to isolate and introduce only one or more desirable genes without introducing undesirable genes into the target organism. Gene Cloning Experiment 1. A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule. 2. The vector transports the gene into a host cell, which is usually a bacterium although other types of living cells can be used. 3. Within the host cell the vector multiplies, producing numerous identical copies, not only of itself but also of the genes that it carries. 4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place. 5. After a large number of cell divisions, a colony, or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule; the gene carried by the recombinant molecule is now said to be cloned. Basic Steps in Gene Cloning + + Vector Fragment or DNA Recombinant DNA molecule Bacterium Bacterium carrying recombinant DNA molecule 2. Transport into the host cell 1. Construction of a recombinant DNA molecule 3. Multiplication of recombinant DNA molecule 4. Division of host cell 5. Numerous cell divisions resulting in a clone Bacterial colonies growing on solid medium Therefore, cloning is making multiple identical copies of any template DNA.
NEET Biotechnology - Principles and Processes 73 Aakash Educational Services Limited - Regd. Office : Aakash Tower, 8, Pusa Road, New Delhi-110005 Ph. 011-47623456 Let us now focus on the first instance of the construction of an artificial recombinant DNA molecule. The construction of the first recombinant DNA emerged from the possibility of linking a gene-encoding antibiotic resistance with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of Salmonella typhimurium. Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance. The cutting of DNA at specific locations became possible with the discovery of the so called ‘molecular scissors’ - restriction enzymes. The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vectors to transfer the piece of DNA attached to it into the host organism. The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in-vitro known as recombinant DNA. When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic resistance gene in E.coli was called cloning of antibiotic-resistance gene in E.coli. Thus, there are three basic steps in genetically modifying an organism: (i) Identification of DNA with desirable genes. (ii) Introduction of the identified DNA into the host. (iii) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny. TOOLS OF RECOMBINANT DNA TECHNOLOGY Tools required to accomplish genetic engineering include : 1. DNA manipulating enzymes (i) Restriction enzymes (ii) Polymerase enzymes (iii) Ligases enzymes 2. Vectors 3. Host organism Restriction Enzymes (RE) In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in E.coli were isolated. One of these added methyl groups to DNA (methylase), while the other cut DNA. The later was called restriction endonuclease. Restriction enzymes serve as chemical knives to cut genes (= DNA) into defined fragments. These may then be used (i) To determine the order of genes on chromosomes. (ii) To analyze the chemical structure of genes and of regions of DNA which regulate the functions of gene. (iii) To create new combinations of genes. The first restriction endonuclease – Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II. The sequence is 5 GT (Pyrimidine : T or C) (Purine : A or G) AC3 3 CA (Purine : A or G) (Pyrimidine : T or C) TG5 Besides Hind II, we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.
74 Biotechnology - Principles and Processes NEET Aakash Educational Services Limited - Regd. Office : Aakash Tower, 8, Pusa Road, New Delhi-110005 Ph. 011-47623456 Note : Restriction enzymes are obtained only from prokaryotes. It is their natural defense mechanism against bacteriophage infection. The convention for naming these enzymes is that the first letter comes from the name of the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated. For example, EcoRI comes from Escherichia coli RY13. In EcoRI, the letter ‘R’ is derived from the name of strain “Rough”. Roman number following the names indicate the order in which the enzymes were isolated from that strain of bacteria. Restriction enzymes belong to a larger class of enzymes called Nucleases. Nucleases Exonuclease Endonuclease (a) An exonuclease (b) An endonuclease Cleavage Cleavage Hydrogen bond Nucleotide Phosphodiester bond Cleavage Fig. : The reactions catalyzed by the two different kinds of nuclease. (a) An exonuclease, which removes nucleotides from the end of a DNA molecule. (b) An endonuclease, which breaks internal phosphodiester bonds. Each restriction enzyme recognises a specific palindromic nucleotide sequence in DNA. A palindrome is a word, phrase, number or other sequence of units that can be read the same way in either direction. Palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept same. These sequences may range from 4–8 nucleotides in length. For example, the following sequences reads the same on the two strands in 5 3 direction. This is also true if read in the 3 5 direction. 5  GAATTC  3 3  CTTAAG  5 Table : Recognition Sequences of Several Restriction Endonucleases Enzyme Bam HI Eco RI Hind III Pst I Sal I Microbial origin Bacillus amyloliquefaciens Escherichia coli Haemophilus influenzae Providencia stuartii Streptomyces albus Recognition site 5 –G–G–A–T–C–C–3   3 –C–C–T–A–G–G–5   5 –G–A–A–T–T–C–3   3 –C–T–T–A–A–G–5   5 –A–A–G–C–T–T–3   3 –T–T–C–G–A–A–5   5 –C–T–G–C–A–G–3   3 –G–A–C–G–T–C–5   5 –G–T–C–G–A–C–3   3 –C–A–G–C–T–G–5   Sma I Serratia marcescens 5 –C–C–C–G–G–G–3   3 –G–G–G–C–C–C–5   Note : There are three types of restriction enzymes though most restriction enzymes employed in genetic engineering are Type II because they can be used in-vitro to recognise and cut within the specific DNA sequence typically consisting of 4–8 nucleotides.