Genetics and Gene Biology

Gene Cloning (rDNA)

Gene cloning is the process of producing a large number of copies of gene or a given DNA sequence. The technique is also known as molecular cloning.

The technique was pioneered by Paul Berg, Herbert Boyer and Stanley Cohen. Gene cloning involves the insertion of target DNA or foreign DNA into suitable vector in such a way that inserted DNA replicates independently and are transferred to progenies during cell division, thereby generating identical organisms. The cloned DNA obtained is called as Recombinant DNA.

Breakage of DNA molecule at two desired places to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position will give a product called recombinant DNA. And the technique is known as recombinant DNA technology/ genetic engineering/gene cloning.

Cloning of the gene has been a common practice in the field of genetics, molecular biology and microbiology. It has been used to create copies of target gene for applications such as gene sequencing, mutagenesis, genotyping, heterologous expression of proteins etc.

Recombinant DNA

The artificial created molecule formed by insertion of foreign DNA into vectors is called recombinant DNA molecule. The creation of artificial recombinant DNA molecule by variety of sophisticated techniques in many cases it is often related as genetic engineering or gene manipulation.

Basic principle

  • Isolation of the DNA sequence (gene) of interest from the genome of an organism or from a gene library. This usually involves DNA purification followed by enzymatic digestion or mechanical fragmentation, to liberate target DNA sequence.
  • Insertion of target (foreign) DNA/gene into DNA molecule (vector) to produce recombinant DNA molecule which is capable of replicating in a host cell. I.e. cloning vector. Suitable cloning vectors for bacterial cells include plasmids and bacteriophages.
  • Introduction of rDNA molecule into a suitable host e.g. E. coli. The process is termed as transformation. When plasmid is used or transduction for a recombinant virus vector.
  • Selection and growth of the transformed cells, using the techniques of cell culture.

 

Requirements

1. Target DNA: DNA segment with the gene of interest is target DNA. It can be obtained by isolation of DNA sequence (gene) of interest from the genome of an organism or from a gene library.

2. Cloning Vectors: Clone vectors are the DNA molecules that acts as a vehicle for carrying a foreign DNA fragment when inserted into it and transports it into a host cell which usually a bacterium and other living cells too.

The ideal vectors used to simplify for the multiplication of DNA or for expression of the cloned genes must have following features:

  1. Capability of autonomous replication
  2. Small size
  3. Presence of selective marker genes.(eg, Antibiotic resistance genes, lac Z or resistance to toxins) allowing easy detection of recombinants
  4. Presence of unique restriction sites or multiple cloning sites for target DNA
  5. Ease of purification
  6. Null effect on the replicative avaibility of vector due to insertion of target DNA.

Vectors used in gene cloning involves Plasmids, Bacteriophage, Cosmids, Shuttle vectors.

 

3. Enzymes: Number of enzymes are involved in cloning of the gene. Major enzymes includes the following

  1. Restriction endonuclease
  2. DNA ligase

 

  • Restriction endonuclease is the enzymes that cut at specific sites within a molecule of DNA. Such sites at which cleavage occurs are called restriction site, while the sequence recognized by the enzyme is called a recognition site.
  • DNA ligase is the enzyme that catalyze ligation or joining up of the DNA sequences. This ATP dependent enzyme is capable of forming covalent phosphodiester bonds between annealed DNA molecules, thereby creating links/ join between DNA fragments.

 

4. Suitable host: Host refers to the organism that takes up the recombinant DNA and replicate it effectively to produce multiple number of copies. E.g. Escherichia coli is extensively used as a host for production of insulin through recombinant DNA technology.

5.Detectants for transformants: Transformants refers to the cells that has undergone the process of transformation by taking up recombinant DNA. Detectants are the substances that are used for identification and selection of transformed cells for further process.

Uses of Gene Cloning

DNA cloning or gene cloning process has been used in various fields such as molecular biology, health and medicine, agriculture etc. Recombinant DNA obtained from cloning are used for various purposes. Some of the uses of them are:

  1. Gene Therapy: It involves an attempt to provide normal copy of gene to cells of body in cases of patients with genetic disorders (They lack functional form of particular gene). E.g. Use of gene cloning to create plasmid containing normal version of gene which is nonfunctional in cystic fibrosis.

 

  1. Gene Analysis: It involves use of recombinant genes to study the function of normal genes. This method is commonly used in research labs for analysis of properties and functions of the target genes. E.g. Study of antibiotic resistant genes in microorganisms.

 

  1. Agriculture: Insertion of recombinant gene / cloning of bacteria has proven to increase the nitrogen fixation.

 

  1. Pharmaceuticals: Gene cloning has been used in production of human proteins such as insulin, growth hormone etc. Tissue plasminogen activator (tPA), used for treatment of strokes as well as prevention of blood clots is produced through recombinant DNA technology.

Gene cloning is also used for productions of various other proteins, vitamins etc.