[SUMAN-SAJAN]

Recombinant DNA technology

• Also known as genetic engineering, molecular biology technology or cloning. The collection of techniques that allow the purification, manipulation and use of genetic material. These techniques are mostly based on the use of enzymes purified from bacteria and animal cells, that allow steps such as DNA copying (replication), conversion into RNA (transcription), and into protein (translation), to be recapitulated in the laboratoryThese steps include specific cutting of DNA at defined sites, joining or splicing of DNA fragments, and numerous other modifications which allow manipulation down to the level of a single base pair. These techniques allow the manipulation of DNA; the conversion of these genes into protein by expression in biological vats such as bacteria, and yeast; the bioengineering of viruses; the production of engineered organisms such as transgenic animals and knockout mice; and the development of DNA-based forensic and diagnostic tests

How recombinant technology works

• Recombinant technology begins with the isolation of a gene of interest. The gene is then inserted into a vector and cloned. A vector is a piece of DNA that is capable of independent growth; commonly used vectors are bacterial plasmids and viral phages. The gene of interest (foreign DNA) is integrated into the plasmid or phage, and this is referred to as recombinant DNA.
• Before introducing the vector containing the foreign DNA into host cells to express the protein, it must be cloned. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells

Restriction enzymes
Restriction enzyme is called molecular biology secissor .





Restriction endonucleus - cut DNA molecules in very specific locations, these are produced by bacteria
Resriction enzymes split DNA at "pallidroms"
Pallidroms = reads the same forward as backward; e.g. radar



• Example - +Hind III restriction endonucleus
splits between A---A

• ---A---A---G---C---T---T---
---T---T---C---G---A---A---

Tools of rDNA technology

The Southern Technique

DNA fragments are transfered to nitrocellulose sheets where they bind.
DNA fragments are denatured and separated by gel electrophoresis
Fragments are blotted onto a sheet of nitrocellulose and fixed by heating
Blot is reacted with a radioactive probe of RNA or DNA which binds to complementary DNA
Autoradiography is used to detect radioactive fragments



Northen Blot for RNA, sourhern blot technique is named on scientist who identified it.
Western Blot for protein.
Restriction Fragment length polymorphisms(RFLPs)
Human genomes is hetrogeneous, containing slight variation is base sequence that differ from individual to individual.
Polymerase chain reaction:amplifying a selected DNA fragments.



Application of r DNA in medical fields
rDNA has also impacted the diagnosis and treatment of diseases and genetic disorders in many areas of medicine. It has enabled many advances, including.
• Isolation of large quantities of pure protein
In addition to the follicle-stimulating hormone (FSH) used in Follistim® AQ Cartridge, insulin, growth hormone and other proteins are now available as recombinant products.

• Identification of mutations
People may be tested for the presence of mutated proteins that may be associated with breast cancer, retino-blastoma, and neurofibromatosis.

Production of insulin by rDNA process


• Diagnosis of affected and carrier states for hereditary diseases
Tests exist to determine if people are carriers of the cystic fibrosis gene, the Huntington’s disease gene, the Tay-Sachs disease gene, or the Duchenne muscular dystrophy gene.
• Transferring of genes from one organism to another (Gene therapy )
: People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and certain cancers may now benefit from the progress made in gene therapy.
• Mapping of human genes on chromosomes
Scientists are able to link mutations and disease states to specific sites on chromosomes.

Parental diagnosis of diseases: Damniotic fluid can be used for predicting the risk of developing genetic diseases e.g. sickel cell anemia.
Transgenic: the somatic gene replecment therapy will not pass to the offspring. Transgenesis refer to the transfer of genes into the fertilized ovum which will be found in somatic as well as ger cells and pass on sucssescive genretion.
Labroratory diagnostic applicaton: by using the rDNA technology, the diagnosis of many disease has become simple and rapid e.g. AIDS.