Recombinant DNA technology class 12th

You ever wondered how researchers make new vaccines for so many diseases ? We do have limited amount of bacteria and there are numerous diseases out there waiting to harm us. Someone must play the role here, right ? That role is played by genetic engineering. 

Genetic engineering means to make changes or modifications in characteristics by controlling its genetic material. To make such changes recombinant DNA technology is used about which we will be reading in this article. 

What is recombinant DNA technology ?

Recombinant DNA technology is a technique by which we can change or make modifications in the characteristics of an organism by controlling its genetic material to make changes in the phenotype of that organism. Recombinant DNA technology is used to make useful characteristics in organisms like having disease resistance ability and also by the help of these modified organisms we can make insulin for humans, hormones, vaccines, enzymes, etc.. 

Though recombinant DNA technology was first constructed by Stanley Cohen and Herbert Boyer in the year 1972, this idea was very first proposed by a graduate student Peter Lobban with A. Dale Kaiser at the Department of Biochemistry, Standford University

Stanley and Herbert first isolated the antibiotic resistant gene from plasmid. They used the bacteria called "Salmonella typhimurium" and linked with another plasmid DNA which was acting as a vector. Now, you must be confused that which does these words "vector" and "plasmid" mean. Don't worry, keep reading further. 

Plasmid: Plasmids are small, extra-chromosomal, double stranded, circular forms of DNA that can reproduce on its own. These are found in bacteria, yeast and sometimes in plant and animal cells. 

Plasmids are considered to be transferable genetic elements or replicons which can replicate on its own when it is inserted into a suitable host. The plasmids in bacteria carry genes which are related to metabolic activity and allows the carrier bacterium to survive and reproduce under unfavourable conditions. 

Vector: Vectors are the molecules of DNA which are used to transfer the genetic materials into another cell. Must be confused right ? For that I have a quick example for you go through in order to understand what a vector is. 

Eg, a mosquito transfers the malarial parasite into human beings that means it acts as an insect vector. 

Now you the meanings of words plasmid and vector we will now be reading about the process of recombinant DNA technology. 

Process of recombinant DNA technology:

The process of recombinant DNA technology is not that tough the way it seems in the books. Like I always say, you need to concentrate and understand the topic in order to know more about it and I always make sure that I make you understand in an easy way. The process of recombinant DNA is as follows:

DNA is taken by both the sources and is cut at specific points by the help of restriction endonuclease. The cut portion is known as the restriction fragment. The cut is made with the help of restriction enzyme at a specific point. Same restriction endonuclease cuts a matching DNA sequence from plasmid as well. Plasmid acts as a vector to transfer the piece of DNA attached to it

Now, the ends of the cut have an overhanging piece of single-stranded DNA which are called the "sticky ends". These sticky ends are able to form base pairs with any DNA fragment containing a complementary sticky end. Both the ends, of the fragment and of the plasmid is linked by the help of enzyme DNA ligase. The fragment is placed in the place of the cut DNA segment of the plasmid and this DNA is known as recombinant DNA

This piece of DNA is now inserted into a host cell. Now, this recombinant DNA needs to be cloned i.e to be made a copy of. There are 2 ways you can do this by 1. By in vitro technique by using the Polymerase Chain Reaction (PCR) or 2. By in vivo technique which is where the recombinant DNA is inserted into the host and it starts to clone inside the cell

In vivo can be done by using unicellular prokaryotes, unicellular eukaryotes or mammalian tissue culture cells. After the recombinant DNA is inserted into the host bacterium the divides to make many copies of the recombinant DNA, these copies are then preserved in a DNA library which is also called genomic DNA library

Process of recombinant DNA technology

Applications of recombinant DNA technology:

Recombinant DNA technology has a lot of uses which is useful to us humans in some way or the other. Uses of recombinant DNA technology are as follows:
  1. Recombinant DNA technology can be used to explain the molecular events that takes place in biological process of cellular differentiation where cells change their type from one form to another or the process of ageing. 
  2. Gene maps can be made precisely. 
  3. Can be used to read out complete nucleotide sequence of genomes of many different organisms even humans. 
  4. By the help of recombinant DNA technology many useful chemical compounds can be produced at a lesser price and there is less wastage of the resources. 
  5. Vaccine is produced for hepatitis B via the help of recombinant DNA technology.

Products produced by the help of recombinant DNA technology:

As in the above section you read the uses of recombinant DNA technology. Now, we will be reading about the actual therapeutic products that have been made by the help of recombinant DNA technology. Following are the products produced:
  1. Blood proteins - Erythropoietin, Factors VII, VIII and IX, tissue plasminogen activator and urokinase. 
  2. Human Hormones - Epidermal growth factor, follicle stimulating hormone, insulin, nerve growth factor, relaxin and somatotropin. 
  3. Immune modulators - α - interferon, β - interferon, colony stimulating factor, lysozyme ad tumor necrosis factor. 
  4. Vaccines - Vaccines for cytomegalovirus, hepatitis B, measles and rabies. 

Tools used in recombinant DNA technology:

Nothing advanced is used in this technique but, if you payed close attention to the process you will know what are the tools which were used in this recombinant DNA technology. Let us now see the tools or you can also call it as materials used for recombinant DNA technology.

1. Enzymes:

Enzymes are the main tools used in recombinant DNA technology without it this whole process wouldn't even be taking place right here. They belong to a larger class of enzymes called nucleases. The enzymes used in recombinant DNA technology are restriction endonuclease, DNA ligase, reverse transcriptase, DNA polymerase, alkaline phosphatases, etc.. All these enzymes have a feature of their own and I would give you a brief knowledge about these enzymes. 

Restriction endonuclease is used to cut the DNA at specific points. They are also called biological/molecular/chemical/scissors/ knives/scaples. There are three main types of restriction endonuclease, which are as follows:

Type 1: These type of restriction endonuclease has two functions i.e, of restriction and of modifications. They have a recognition sequence of about 15 base pairs. As this causes cleavage of DNA at the sites which are non specific (1000 base pairs away from recognition sequence) these type of enzymes cannot be used in recombinant DNA technology. 

Type 2: This type of restriction endonuclease can be used in vitro technique has it recognizes as well as splits inside specific DNA sequences. They have a recognition sequences of about 4-8 base pairs. As they cause cleavage at unmethylated site inside their recognition sequences it can be used in recombinant DNA technology

Type 3: This type of restriction endonuclease comes in between type 1 and type 2 restriction endonuclease. It is also site-specific but had different requirements than the other. This type also has two functions, i.e, of restriction and of methylation. Hence, it cannot be used in recombinant DNA technology. 

The enzyme ligase is used to join both of the ends of the DNA to the plasmid. Reaction of ligase which is called ligation needs a source of energy to join the ends of the DNA to the plasmid. 

The enzyme DNA Polymerase is used for the synthesis of the complementary DNA strands on DNA template where as reverse transcriptase is used for synthesis of the complementary double stranded DNA strand on single stranded RNA template.

 2. Cloning vectors:

 Vectors are the molecules of DNA which are used to transfer the genetic materials into another cell. They have a low molecular weight of DNA molecules which makes it easier to multiply independently of the genomic DNA. 

Vectors can be used to make several copies of the DNA fragment you wish of by the help of division. It is necessary for vectors to have replication origin because it has to be able to make copies in the host cell. Following are the six types of cloning vectors used in recombinant DNA technology:
  • Plasmids
  • Bacteriophages
  • Plant/animal viruses
  • Transpons
  • Artificial chromosomes of bacteria, yeast and mammals. 
  • Cosmid
Out of the following vector, plasmids and viruses are the most commonly used vectors. The plasmids which are used as vectors at present are engineered in such a way that they help to easily lik the foreign DNA and selects recombinant from non-recombinants. 

Vectors requires a selectable marker which helps in identification and elimination of non-transformed cells. It simply reduces the growth of the transformed cells.

3. Target DNA or foreign DNA:

The DNA which will be used in the vector apart from the original segment of DNA is called as target DNA or foreign DNA. This type of DNA is also called as desired gene. 

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