DNA Fragments

Página 1

DNA Fragments

A number of diseases result from organisms being unable to produce metabolic chemicals for themselves, most common of which is insulin. This is due to a specific portion of DNA, they are the product of a gene. Previously, the only way to treat such diseases was to extract the required chemical from other organisms and inject them into the patient. The donor would typically be an animal or a human. However, problems such as rejection by the immune system and infection arise when this method is taken. The cost is also considerable. The solution to this is producing large quantities of 'pure' proteins from other sources, such as bacteria. In order to do this, methods were developed to isolate, clone and transfer genes into micro-organisms (bacteria). These micro-organisms then reproduce asexually through mitosis and replicate the inserted gene, essentially creating a "gene factory". Since the gene is continuously replicated, so is the protein that the gene codes for. Combining DNA from two separate organisms produce Recombinant DNA. The resulting organism is a Genetically Modified Organism (GMO).

Inorder to produce the desired protein via DNA technology, various steps of gene transfer and cloning must be taken. This involves:

Isolation of the DNA fragments that contain the gene that codes for the desired protein

Insertion of the DNA fragments into a vector (e.g. bacteria)

Transformation - the transfer of DNA into suitable host cells

Identification of the host cells that have successfully accepted the gene through use of gene markers

Growth/Cloning of the population of host cells

There are two main enzymes that are used to isolate genes. These enzymes are Reverse Transcriptase and Restriction Endonuclease. Reverse transcriptase is used to produce DNA from RNA. First, a cell that produces a large quantity of the mRNA that codes for the desired protein is selected. Since the cell produces a large quantity of the desired mRNA, some is extracted. Free nucleotides then bind to the complimentary nucleotides on the mRNA strand. Reverse transcriptase then binds these free nucleotides when they are in close proximity due to binding to the mRNA strand. This produces a single strand of Complimentary DNA or cDNA for short. The cDNA then detaches from the mRNA. Free nucleotides then bind to the cDNA. DNA Polymerase then binds these free nucleotides, forming a Double Stranded DNA molecule. This is a copy of the gene that codes for the desired protein.

There is more than one type of Restriction Endonuclease. However each type has one thing in common, they cut the DNA double strand at a specific sequence of bases known as a Recognition Sequence. Sometimes, this cuts between two base pairs, leaving two straight edges known as Blunt Ends. The other way in which Restriction Endonuclease cuts the DNA strand is in a staggered fashion. This leaves "over hanging" edges, with exposed and unpaired bases. For this to occur, the Recognition Sequence must be the same but opposite (e.g. the top strand is AAGCTT and the bottom strand is TTCGAA). This is called a Palindrome. When the Restriction Endonuclease cuts like this, the staggered ends left are known as Sticky Ends.

New Page