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Biotechnology: Principles and Processes

Tools of Biotechnology

What comes to your mind when you hear the term ‘Biotechnology’? Is it the process of making curd or bread using microorganisms? Or is it the process of using gene therapy to correct a defective gene? You will be surprised to know that both the above processes fall under biotechnology! Let’s learn more about the different tools of biotechnology.

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Principles Of Biotechnology

The field of biotechnology has evolved tremendously over the years. It has gone from the traditional view of using microbes to generating products for human use, to the modern view of using genes for developing vaccines. Therefore, the European Federation of Biotechnology (EFB) has come up with a new definition that involves both the traditional and the modern view of biotechnology. It states that –

‘Biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogs for products and services’. The two core techniques responsible for the birth of modern biotechnology are:

  • Genetic Engineering: It involves techniques that change the chemistry of the genetic material such that when they are introduced into the host, the host phenotype changes.
  • Maintenance of a contamination-free environment to enable the growth of only the desired microbe in large quantities during the manufacture of products like vaccines, antibiotics etc.

Genetic Engineering

Traditional hybridization methods in plant and animal breeding introduce desirable as well as undesirable genes. The techniques of genetic engineering such as recombinant DNA technology, gene cloning etc. overcome this problem, allowing us to introduce only desired genes. Genetically modifying an organism has the following basic steps:

  • Identification of DNA containing desired genes.
  • Introduction of desired DNA into the host organism.
  • Maintenance of the introduced DNA in the host genome and transfer of the DNA to its progeny.

Let us now learn about the tools of biotechnology.

Tools Of Biotechnology

Genetic engineering or recombinant DNA technology is possible only if we have some important tools. The following are some important tools of biotechnology.

1) Restriction Enzymes

Restriction enzymes, also known as ‘restriction endonucleases’ are molecular scissors that can cut DNA at specific locations. These are part of a larger class of enzymes called ‘Nucleases’. Nucleases are of two kinds:

  • Exonucleases – Enzymes that remove nucleotides from the ends of DNA.
  • Endonucleases – Enzymes that make cuts at specific positions in the DNA.

Hind II‘ – the first restriction endonuclease to be isolated, identifies a specific six base pair sequence and always cuts the DNA at a particular point. This specific sequence is the ‘Recognition sequence‘. Today, there are more than 900 restriction enzymes, isolated from about 230 bacterial strains. Each of these enzymes recognizes different recognition sequences. There is a naming convention to name restriction enzymes. Let’s learn this using EcoRI as an example:

  • Eco – indicates that this enzyme was isolated from Escherichia coli.
  • R – refers to the name of the strain.
  • I – is the Roman numeral that indicates the order of isolation of this restriction enzyme from the strain of bacteria.

Mode of Action of Restriction Enzymes

Endonucleases perform their function in the following manner:

  • Inspect the length of DNA for the specific recognition sequence.
  • Bind the DNA at the recognition sequence.
  • Cut the two strands of DNA at specific points in their sugar-phosphate backbone.

Restriction enzymes recognize specific palindromic nucleotide sequences in the DNA. What is a palindrome? In terms of words, a palindrome is a group of letters that produce the same word when reading forward or backwards. An example is ‘RADAR’. In terms of DNA, a palindrome is a sequence of base pairs that reads the same on both DNA strands when reading in the same orientation. For example –

5′ —— GAATTC —— 3′
3′ —— CTTAAG —— 5′

On reading both the strands shown above in the 5′ to 3′ direction, they give the same sequence. This is true even when they are both read in the 3′ to 5′ direction.

Restriction enzymes cut the DNA strand a little away from the center of the palindromic site, but between the same two bases on both strands. This gives rise to single-stranded, overhanging stretches on each strand called ‘sticky ends’. They are called ‘sticky’ because they can bind to their complementary cut counterparts.

Using restriction enzymes we can generate recombinant DNA that contains DNA from different sources. Cutting these DNA sources with the same restriction enzyme gives fragments with the same kind of ‘sticky ends’, that can then be joined using DNA ligases.

Tools of Biotechnology

Mode of action of EcoRI [Source: Wikimedia Commons]

2) Cloning Vectors

Just the way a mosquito acts as a ‘vector’ to transfer the malarial parasite into the human body, we need vectors to transfer the cut DNA into a host organism. Vectors are one of the important tools of biotechnology.

Plasmids make good vectors because they can replicate in bacterial cells, independent of the control of the chromosomal DNA. The vectors in use currently are engineered such that they help in easy linking of foreign DNA and allow selection of recombinants over non-recombinants. A vector needs the following features to enable cloning:

(i) Origin Of Replication (ori)

This is the sequence from where replication begins. Linking a piece of DNA to this sequence causes it to replicate in the host cell. This sequence also controls the copy number of the linked DNA. Therefore, the target DNA needs to be cloned into a vector whose ‘ori’ supports high copy number, in order to recover large amounts of the DNA.

(ii) Selectable Marker

The vector also needs to have a selectable marker which allows the selection of recombinants over non-recombinants. In terms of E. coli, some useful selectable markers are genes that provide resistance to antibiotics like ampicillin, kanamycin, chloramphenicol etc. Since the normal E. coli cells do not carry these resistance genes, it becomes easy to select the recombinants.

(iii) Cloning Sites

In order to attach the foreign DNA to a vector, the vector should have a recognition site for a specific restriction enzyme. Multiple recognition sites will result in multiple DNA fragments, complicating the process of cloning. A vector has more than one antibiotic resistance gene. The foreign DNA is ligated into a restriction site in one of the antibiotic resistance genes.

For example, let’s say an E. coli cloning vector has genes for ampicillin and tetracycline resistance. On ligating the foreign DNA into a recognition site within the tetracycline resistance gene, the plasmid loses its tetracycline resistance. But, it can still be selected for non-recombinants by plating on ampicillin-containing medium.

Now, by transferring the ones that grow in the ampicillin medium to a medium with tetracycline, we can dissect out recombinants from non-recombinants. The recombinants will grow in ampicillin but not in tetracycline medium; while non-recombinants will grow in both mediums.

Currently, alternative markers are available that can differentiate recombinants from non-recombinants based on their ability to produce color. This involves insertion of the foreign DNA in the DNA sequence of an enzyme like β-galactosidase, which inactivates the enzyme. This is ‘insertional inactivation’. On reaction with a substrate, the recombinants do not produce color whereas non-recombinants produce color.

Tools of Biotechnology

Blue-white screening using B-galactosidase [Source: tankonyvtar]

(iv) Vectors to Clone Genes in Plants and Animals

Long before us, bacteria and viruses knew how to transfer genes into plants and animals. For example, Agrobacterium tumifaciens, a pathogen on dicot plants, transfers ‘T-DNA’ that transforms normal plants cells into tumours. These tumours then produce chemicals that the pathogen requires. With better understanding, we have now converted these pathogens into useful vectors to deliver genes of interest to the plants or animals.

Now, the tumour-inducing (Ti) plasmid of Agrobacterium tumifaciens has been modified into a cloning vector. This vector is no longer harmful but is useful in delivering genes of interest to plants. Retroviruses transform normal cells into cancerous cells in animals. These have also been modified so that they are no longer harmful and can deliver genes to animals.

3) Competent Host

Host cells are bacterial cells which take up the recombinant DNA. Since DNA is hydrophilic, it cannot pass through the cell membrane of bacteria easily. Therefore, the bacterial cells have to be made ‘competent’ to take up the DNA.

Some procedures that make the cells competent are treatments with a specific concentration of divalent cation like calcium. This makes it easy for the DNA to enter the cell wall through pores. Incubation of cells with the recombinant DNA on the ice, followed by heat shock at 42°C and another incubation on ice, enables the cells to take up the DNA.

There are several other methods to introduce foreign DNA into host cells. The ‘microinjection’ method involves injecting the recombinant DNA directly into the nucleus of an animal cell. The ‘bolistics’ or ‘gene gun’ method bombards plant cells with high-velocity microparticles of gold or tungsten coated with DNA. The last method uses ‘disarmed pathogen vectors’ (discussed above) to transfer the recombinant DNA into the infected host cells.

Solved Example For You

Q1: Which of the following sequences is not palindromic?

  1. 5'GGATCC 3'
    3'CCTAGG 5'
  2. 5'AGCT 3'
    3'TCGA 5'
  3. 5'TGCT 3'
    3'TCGA 5'
  4. 5'TCGA 3'
    3'AGCT 5'

Sol: The correct answer is the option ‘c’. The sequence on the two strands is not the same when both are read in the same orientation i.e. in 5′ to 3′ direction or 3′ to 5′ direction.

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