Recombinant DNA technology

Introduction

     Recombinant DNA technology is a series of procedures that are used to join DNA segments from different sources. This is in vivo method which is used when gene cloning is required at an industrial scale. The main advantage of this method is the production of a product of genes beside copies of the gene. It involves the selection and isolation of the desired gene, inserting it in a suitable vector, and the transformation of a suitable host by the recombinant DNA.



Tools or components of recombinant DNA technology

     The cloning of gene through recombinant DNA technology requires the following components or tools
  1. Gene of interest
  2. Molecular scissors
  3. Molecular carrier or vector
  4. Molecular glue
  5. Expression system

1. Gene of interest

   The gene of interest is the gene that is to be cloned. It can be obtained by one of the three possible ways;
  1. Artificial gene synthesis is the process of synthesizing a gene in vitro without template DNA samples with the help of a DNA synthesizer machine.
  2. Gene of interest can also be obtained by synthesizing it from its mRNA. Synthesis of the gene from mRNA is carried out by reverse transcriptase enzymes which are naturally found in retroviruses. The DNA formed by this process is called complementary DNA (cDNA).
  3. In most of the cases, the gene of interest is directly cleaved from a chromosomal DNA by using particular DNA scissors called restriction endonucleases.

2. Molecular scissors (Restriction endonucleases)

       Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of duplex DNA at specific sequences. In 1970, Hamilton O. Smith at Johns Hopkins University, isolated the first restriction enzyme. Many different restriction endonucleases have been isolated so far.
     Naturally, restriction enzymes are found in bacteria, where they appear to serve as a host-defence role because they chop up and inactivate the DNA of infecting viruses.
     Each restriction enzyme cleaves DNA at a specific sequence of DNA called recognition sites or restriction sites. These sites have palindromic sequences. A palindromic sequence is a four to eight base pairs in DNA in which nucleotides are arranged symmetrically in reverse order.
     Restriction enzymes either make a staggered cut or blunt cut. A staggered cut is one in which the resulting duplex fragments show single-stranded projected ends called sticky ends. While in blunt cut the resulting duplex fragments do not show sticky ends.

3. Molecular carriers or vectors

     Vectors are another major component required to make a recombinant DNA (rDNA) molecule for gene cloning. Vectors act as a vehicle for carrying foreign DNA into a host cell for multiplication. Usually, small circular DNA molecules of bacterial origin are used as cloning vectors. A DNA molecule should possess the following essential characteristics to act as a cloning vector.
  • Origin of a replication site.
  • Antibiotic-resistant genes.
  • Multiple cloning sites (MCS) or the poly-linker which contain restriction sites of different enzymes.
For example Plasmid, lambda phage DNA, Cosmid (the combination of plasmid and phage DNA), Yeast artificial chromosomes (YACs), etc.

4. Molecular glue (DNA ligase)

    This enzyme is responsible for the formation of the phosphodiester linkage between two adjacent nucleotides and thus joins two double-stranded DNA fragments; therefore it is called molecular glue. In rDNA experiments, DNA ligase is used to join two different DNA fragments (plasmid/vector and the foreign DNA) that are annealed by sticky ends.

5. Expression system

     A suitable organism that can acts as a host for the recombinant vector to express (multiplication) is called an expression system. Therefore, the selection of a suitable expression system always depends upon the type of vector which is being used while making recombinant DNA. The most important characteristics of an ideal expression system is its short generation time and simplicity of its genetic system. So bacterial cells can act as an ideal expression system.

Mechanism or procedure

    Cloning of the desired gene through recombinant DNA technology involves the formation of recombinant DNA (gene of interest + vector DNA), the transformation of a suitable expression system by the recombinant DNA, and the identification of transformed clones.

Formation of recombinant DNA

   The first step in the construction of a recombinant DNA is the isolation and purification of vectors and the DNA fragments containing the gene to be cloned. First, digest the vector DNA (e.g., plasmid) with a suitable restriction so that sticky ends can be produced. Next, isolate the DNA fragment carrying the gene of interest with the help of the same restriction enzyme. Now both, vector and foreign DNA are inoculated together in the presence of DNA ligase which connects them by forming phosphodiester linkage. This results in the formation of a recombinant DNA molecule of vector and the gene of interest.

Transformation of expression

       Here transformation refers to the insertion of recombinant DNA into the expression system which can be performed by putting the expression system (bacterial cells) and recombinant plasmids into the same medium. Bacterial cells take up the recombinant plasmid, especially, if they are treated with calcium chloride to make them more permeable. Therefore, as the cell reproduces, bacterial clone forms and each new cell contains at least one plasmid. Therefore, each of the bacteria contains the gene of interest, which will express itself and make its product.

Identification of transformed clone

    The transformed clone can be identified by adding a particular antibiotic (for which resistant gene is found in plasmid) into the medium. As the transformed clone has got resistance against the antibiotics, so it remains alive and continues to grow, whereas all the untransformed clones are killed by the antibiotic. From this transformed bacterial clone, the clone gene can be isolated for further analysis or its protein product can be separated.

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