The overall goal of recombinant-DNA technology is to identify, isolate. manipulate. and re-express genes from a given host

Some of the practical goals of such cut-and-paste technology is to:

1) develop a basic understanding of the function and regulation of known gene products.

2) identify new genes whose protein products have not been isolated .

3) correct endogenous genetic defects

4)express foreign genes in disease-susceptible hosts

5) manufacture large quantities of a protein product for widespread use

DNA is composed of two antiparallel strands bound by hydrogen bonds between their nitrogenous-base side chains

Genetic information is provided by the purine and pvrimidine bases which are linked to a sugar phosphate backbone.

This structural support is a series of deoxyribose residues linked by phosphodiester bonds, which are created by covalently joining a hydroxvl group at the 3-carbon position on one deoxyribose residue with a phosphate group located on the 5’ carbon of an adjacent sugar group.

RECOMBINANT DNA TECHNOLOGY: A SERIES OF PROCEDURES USED TO JOIN

TOGETHER (RECOMBINE)DNA SEGMENTS.

A RECOMBINANT DNA MOLECULE IS CONSTRUCTED (RECOMBINED) FROM SEGMENTS FROM 2 OR MORE DIFFERENT DNA MOLECULES. UNDER CERTAIN CONDITIONS, A RECOMBINANT DNA MOLECULE CAN ENTER A CELL AND REPLICATE THERE, AUTONOMOUSLY (ON ITS OWN) OR AFTER IT HAS BECOME INTEGRATED INTO A CHROMOSOME.

An over view

Treat DNA from both sources with the same restriction endonuclease (BamHI in this case).

BamHI cuts the same site on both molecules 5' GGATCC 3'

3' CCTAGG 5' The ends of the cut have an overhanging piece

of single-stranded DNA. These are called "sticky ends" because they are

able to base pair with any DNA molecule containing the complementary sticky end.

In this case, both DNA preparations have complementary sticky ends and thus can pair with each other when mixed.

DNA ligase covalently links the two into a molecule of recombinant DNA.

To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc.

Producing many identical copies of the same recombinant molecule is called cloning.

Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR).

The use of cloning is interrelated with Recombinant DNA in classical biology, as the term "clone" refers to a cell or organism derived from a parental organism, with modern biology referring to the term as a collection of cells derived from the same cell that remain identical.

IN THE CLASSICAL INSTANCE, THE USE OF RECOMBINANT DNA PROVIDES THE INITIAL CELL FROM WHICH THE HOST ORGANISM IS THEN EXPECTED TO RECAPITULATE WHEN IT UNDERGOES FURTHER CELL DIVISION, WITH BACTERIA REMAINING A PRIME EXAMPLE DUE TO THE USE OF VIRAL VECTORS IN MEDICINE THAT CONTAIN RECOMBINANT DNA INSERTED INTO A STRUCTURE KNOWN AS A PLASMID.

Plasmids are extrachromosomal self-replicating circular forms of DNA present in most bacteria, such as Escherichia coli (E. Coli), containing genes related to catabolism and metabolic activity, and allowing the carrier bacterium to survive and reproduce

These genes represent characteristics of resistance to bacteriophages and antibiotics and some heavy metals, but can also be fairly easily removed or separated from the plasmid by restriction endonucleases, which regularly produce "sticky ends" and allow the attachment of a selected segment of DNA, which codes for more "reparative" substances, such as peptide hormone medications including insulin, growth hormone, and oxytocin.

Diabetic patients, whose elevated sugar levels (see fig. 1) are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of abattoir animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans,(2) regulates the use and storage of food, particularly carbohydrates.

Humulin is synthesized by inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce an insulin that is chemically identical to its naturally produced counterpart. This has been achieved using Recombinant DNA technology.