8 Biotechnology- Biology for Human Welfare
8.1 R DNA Technology
R DNA Technology
Dr V Malathi
What is r DNA ?
Recombinant DNA molecules are hybrid DNA molecules formed by joining the DNA sequences /segments from varied sources . Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is
incorporated into a single recombinant molecule. Genetic engineering involves the use of recombinant DNA technology.
Molecular cloning is a set of methods used to construct recombinant DNA and incorporate it into a host organism.
If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic.
Applications of r DNA
- Recombinant DNA (rDNA) is widely used in biotechnology, medicine and research
- Recombinant DNA technology has been used to produce various human proteins in microorganisms
• Examples of products of recombinant DNA technology in medicine and research include: human recombinant insulin, growth hormone, blood clotting factors, hepatitis B vaccine, diagnosis of HIV infection.
• Examples of products of recombinant DNA technology in agriculture include: herbicide-resistant crops, and insect-resistant crops.
What are cloning Vectors ?
Cloning vectors are vehicles that are used to introduce foreign DNA into host cells, where that DNA can be reproduced (cloned) in large quantities.
• Examples of cloning vectors are : plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast
artificial chromosomes (YACs).
What are Tools of r DNA Technology?
Recombinant DNA requires 3 key molecular tools:
1.Cutting DNA at specific sites – most often performed by enzymes called restriction endonucleases (restriction enzymes).
• Restriction enzymes often make staggered cuts at specific 4, 6, or 8-bp palindromic sequences in duplex DNA, leaving characteristic “sticky ends” that can anneal to each other via hydrogen bonding between complementary bases on the single-stranded overhangs.
2.Ligating DNA fragments with an enzyme called DNA ligase.
• DNA ligase, creates covalent phosphodiester bonds between any two DNA fragments that have been cut by the same restriction enzyme, or have the same compatible “sticky ends”.
3.A “vector”, such as a plasmid, that can be used to insert a new segment of DNA via restriction enzyme cutting and ligation. The plasmid containing the inserted DNA segment will replicate in host cells.
What are steps to construct a r DNA?
“Recombinant DNA” by Angela N.H. Creager,creativecommons.org via Wikimedia Commons is licensed under CC BY 4.0
Step 1: Isolation of Gene of interest
Gene of interest is first isolated. For this, initially the cells containing the gene of interest is isolated and disrupted to release nucleus. From the nuclear fraction, the gene of interest is released by using the restriction enzyme which posses the appropriate restriction sites at both ends of the gene of interest. After the gene of interest fragmented, they are separated by using normal iso