This has brought about a plethora of changes in the evolution of laboratory techniques, tools and a focus on platform technologies. To understand these changes it is necessary to gather basic knowledge about what goes on in the laboratory.
A process of biotechnology is called genetic engineering, which is a combination of DNA sequences producing recombinant proteins as eventual therapeutics. This process utilizes restriction enzymes (endonucleases) in bacteria. These enzymes cuts viral DNA into small nonfunctional pieces, thus protecting the bacterium from any invading virus.
There are hundreds of restriction enzymes, each recognizing a specific DNA called a restriction site. EcoR1, a restriction enzyme found in E. coli identifies and cuts at the six-base sequence GAATTS. HaeIII, a restriction enzyme found in Haemophilus-aegyptius, identifies and cuts at the four base sequence GGCC.
Note: [More than 3,800 restriction enzymes have been identified by scientists and 600 are commercially available in the market place.]
Irrespective of its origin DNA is made up of the same four bases i.e. As, Ts, Gs. and Cs. HaeIII detects any DNA segment and cuts it every time it encounters sequence GGCC. The key characteristics of restriction enzymes are that it is specific and reproducible. This helps scientists to utilize restriction enzymes for manipulating DNA.
The opposite of cutting is pasting. The sealing of two DNA segments is achieved by DNA ligase a protein enzyme by a process called ligation. This ability to cut and paste DNA is the premise on which genetic engineering is based.
Note: [Recombinant DNA technology and the discovery of restriction endonucleases earned the noble prize of 1978 to scientists Daniel Nathans, Werner Arber and Hamilton Smith.]
When segments of DNA are cut and pasted together whereby a new DNA called recombinant DNA emerges, and this can be introduced into cells to produce new cells. These cells possess new characteristics, and this genetic alteration can include a single-base letter change or multiple gene changes. The change in a host cell is done by a VECTOR, which physically carry the DNA. A host cell can comprise of bacteria, yeast, plant, insect of mammalian.
Common bacterial vectors include plasmids and phages. A plasmid is s DNA of circular proportion which can engineer and carry a gene of interest. A phage is a genetically engineered virus that injects DNA into bacteria. Cells containing recombinant DNA, are usually referred to as genetically modified transgenic or transformed cells, the process is commonly known as transformation.
Note: [1982, human insulin was approved by FDA in the United States, and this is an instance of the first medicine via recombinant DNA technology]
Recombinant DNA is used in the manufacturing of Recombinant Proteins. The host cell utilizes DNA information and its inherent cell machinery to produce the encoded protein. When these proteins are used as human therapeutics, the host cells must produce and grow in large proportion in order to meet demand. This protein (recombinant protein) is isolated, purified and verified for quality and activity before its marketability.
Producing a protein with proper chain of amino acids entails intense processing in order to be functional and be active. A significant number of human proteins are glycosylated, which signifies that a particular pattern of sugar molecules are linked to them. If translated and not glycosylated then there is the possibility of its malfunctioning. Addition of phosphate group knows as phosphorylation allows the protein to become active. A wilder range of functions get enhanced by addition of other biochemical groups.
Recombinant Proteins include vaccines, hormones, monoclonal antibodies, and hematopoietic growth factors for treating patients of cancer, AIDS, allergies, asthma and a host of different conditions. Due to advanced technology in recent times the number of recombinant proteins have increased significantly.
This is a technique of growing cells in controlled conditions in a laboratory. Transformed bacteria cells and transformed animal cells are processed in order to manufacture recombinant protein drugs. Simple proteins are produced involving DNA technology in bacterial cell cultures, more complex proteins that are glycosylated are formulated in animal cell cultures.
In the process of cell culture, cells are grown in petri dishes or flasks that contain liquid media. This provides nutrients for cell growth, and the cultures are grown in an incubator which maintains proper temperature and environment. Gas mixtures of oxygen and carbon dioxide are often required. The maintenance of specific conditions is a must as any variation will have negative impact in the product produced. Commercial production of proteins have to be sufficient to meet demand. Since there are limitations in growing cells in petri dishes or flasks, it is commercially more viable to transfer cell cultures to larger vessels called bioreactors. Biotechnology scientists have the facility of various state-of-art laboratory equipment in their quest to further the cause of genetic engineering.
This is a machine that replicates DNA which results in Polymerase chain reaction (PCR). PCR is a series of cycles utilizing a minimal amount of original DNA in order to copy and amplify it. Each three step cycle doubles the amount of DNA present and is like a photocopier for DNA. A single piece can churn out million copies of the same DNA. PCR enables scientists with sufficient DNA for their laboratory work. There are various applications for PCR including DNA sequence production, gene for use in creating recombinant proteins.
Note: [For invention and development of Polymerase chain reaction, the noble prize in chemistry was given to Kary Banks Mullis in 1983.]
For analyzing DNA fragments this technique is commonly used. This technical apparatus helps in separating DNA fragments within a gel by allowing electricity to run through it. The fragment is negatively charged and thereby shift to the positive pole of the gel apparatus. The larger DNA fragments move slowly than the smaller ones as they receive resistance from the matrix gel.
There are various types of electrophoresis and separation of gel being one type and types of molecules being the other. DNA, RNA and proteins can all be separated using this method with the relevant apparatus. It has many applications in both clinical and research labs and is generally applied for verification of PCR products. It can therefore be used to cross-check on whether the reaction emanates from the correct DNA fragment.
This is a gene chip which is a small piece of glass or silicon divided into thousands of sections in a grid pattern. Each section has a single-stranded gene fragment corresponding to either a healthy or diseased gene. An individual’s DNA is separated into single strands and attached to a fluorescent dye and then washed over the microarray. The DNA binds to any complementary DNA on the slide and if the sequence are present it becomes a double stranded DNA. The use of computer helps to locate and measure spots on the double stranded fluorescent tagged DNA. Each DNA must match the gene fragment exactly and bind, to indicate the presence of a healthy or diseased DNA. Microarrays are a potent tool for analysis of genes. They are used in genetic testing and, for comparative study of genetic information of individual or species and discovery of drugs.
Researchers are using microarrays in identifying genes and with recombinant DNA technologies. Other microarrays include protein, tissue, chemical compounds and antibody. Thousands of data points are analyzed at a time.
Note: [Microarrays are capable of generating enormous data so that special storage facility is required. There are both public and private microarray data bases]