Monday, 29 February 2016

Process Of Recombinant DNA Technology (Genetic Engineering)

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Recombinant DNA Technology Definition:

Recombinant DNA is the technology used for making artificial DNA (genetic modification) by combining different genetic materials (DNA) from different sources. It is the laboratory methods of genetic recombination to get genetic material (DNA) from different sources which would create a genomic sequence that would not otherwise be found in the genome. 

Gene Cloning Process:

The production of exact identical copies of a particular gene (DNA sequence) extracted from an organism. Identical twins are the perfect example of Gene Cloning.
Gene cloning produces many identical copies of a particular extracted gene. Recombinant DNA technology (genetic engineering) is used when large number of genes is required. The polymerase chain reaction (PCR) is used to create a lesser number of copies within a laboratory test tube.

Process of Recombinant DNA Technology:

Recombinant DNA technology popularly known as genetic engineering aims at synthesizing recombinant DNA also known as recombinant deoxsyribonucleicacid which contains DNA from two different sources. In order to produce recombinant DNA, following are required:

  • Gene of interest, which is to be cloned
  • Molecular scissors, to cut out the gene of interest
  • Molecular carrier (vector), on which gene of interest could be placed
  • An expression system, on which the gene of interest along with the vector is then introduced as a result of which a specific product is made

How to get a gene:

In the process of recombinant DNA technology, first of all we’ll have to get a gene which is to be cloned. There are two ways to get the gene of interest.

  • To Isolate It from The Chromosome: Genes can be isolated from the chromosomes by cutting the chromosomes on the flanking sites of the gene using special enzymes known as restriction endonucleases.
  • Chemical Gene Synthesis by Messenger RNA (mRNA): If the genes are small or for some reason we can’t isolate from chromosomes, they can also be synthesized chemically in the laboratory. To make a gene chemically in laboratory, messenger RNA (mRNA) is used. We’ll use the reverse transcriptase enzyme method. Reverse transcriptase is the enzyme which forms DNA from an RNA template in reverse transcription. This DNA molecule is called complementary DNA (cDNA) and is mainly associated with retro viruses.  

Molecular Scissors (Restriction Endonucleases):

Using of Molecular Scissors is the second step in making recombinant DNA. Molecular Scissors also known as Restriction Endonuclease is the enzyme which cleaves DNA molecules at specific base sequences producing small gene fragments by breaking internal covalent bonds linking nucleotides, used in recombinant DNA technology and chromosome mapping. They are natural enzymes of bacteria, which they use for their own protection against viruses. The restriction enzyme (Restriction Endonuclease) cuts down the viral DNA, but does no harm to the bacterial chromosome. They are called restriction enzymes or molecular scissors because they restrict the growth of viruses. In 1970, Hamilton D. Smith, at Johns Hopkins University, isolated the first restriction enzyme.

Palindromic Sequences:

A palindromic sequence is the nucleic acid sequence (DNA or RNA) which reads the same in both directions. These sequences are recognition sites for enzymes. Bacteria produce a variety of such restriction enzymes, which cut the DNA at very specific sites characterized by specific sequence of four to six nucleotides arranged symmetrically in the reverse order. Such sequences are known as palindromic sequences. So far more than 400 such enzymes have been isolated out of which about 20 are frequently used in recombinant DNA technology.

EcoR1 (A Restriction Enzyme): EcoR1, a commonly used restriction enzyme (endonuclease), cuts double stranded DNA when it has this sequence of bases at the cleavage site. Notice there is now a gap into which a piece of foreign DNA can be placed, if it ends in bases complementary to those exposed by restriction enzyme.

Sticky Ends:

The single stranded but complementary ends of the two DNA molecules are called sticky ends because they can now bind by the complementary base pairing. They therefore facilitate the insertion of foreign DNA into vector DNA.

Molecular Carrier (Vector):

A vector is the mean by which recombinant DNA is introduced into a host cell. One common type of vector is plasmid. Plasmids were discovered by scientists studying the sex life of the intestinal bacterium Escherichia coli.

Plasmids are natural extra chromosomal circular DNA molecules which carry genes for antibiotic resistance and fertility etc. One of the plasmids discovered earlier is PSC 101 has antibiotic resistance gene for tetracycline, whereas PSR 322, has antibiotic resistance gene for tetracycline as well as ampicillin. Inserting gene of interest in tetracycline gene of plasmid (PSR 322) would enable separating out colonies of bacteria in a medium containing ampicillin and vice versa.

Recombinant DNA Preparation:

For preparation of recombinant DNA, the plasmids are cut with the same molecular scissors (restriction enzymes), which was used for isolation of the gene. The gene of interest for instance insulin is then joined with the sticky ends produced after cutting the plasmids with the help of another special enzyme called DNA ligase. DNA ligase is a very important enzyme in recombinant DNA technique that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It seals the foreign piece of DNA into vector. Now the two different pieces of DNA have joined together which is now known as Recombinant DNA or Chimaeric DNA.

Expression of Recombinant DNA:

A clone can be a large number of molecules (i.e. cloned genes) or cells (i.e. cloned bacteria) or organisms that are identical to an original specimen. Bacterial cells take up recombinant plasmid, especially if they are treated with calcium chloride to make them more permeable. Thereafter, as the cell reproduces, a 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 a product. From this bacterial clone, the cloned gene can be isolated for further analysis or protein product can be separated. Besides plasmids, the DNA of bacterial viruses (for example lambda phage) can also be used as a vector. After lambda phage it attaches to a host bacterium, recombinant DNA is released from the virus and enters the bacterium. Here it will direct the reproduction of many more viruses. Each virus in bacteriophage clone contains a copy of the gene being cloned. 

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