The Technique of Manipulating Dna

Genetic engineering is a set of molecular biology techniques that aim at manipulating an organism’s DNA to achieve a particular aim. As Biology Online notes in its definition of the term, genetic engineering can be applied to a number of fields for a number of outcomes, including laboratory techniques (such as recombinant DNA and transgenics), agriculture (e.g., genetically modified foods), and medicine (e.g., the production of drugs such as synthetic insulin, and the isolation of antigen sequences for vaccine production). How genetic engineering works can be broken down into common basic tasks used in all applications: recombinant DNA, cloning, and transformation.

Choosing a DNA Sequence

Before any genetic engineering can occur, the researchers or technicians have to decide on the sequence that will be manipulated. Is it human DNA? Plant DNA? Which vector will be used? The genomic sequence of the organism generally has to be known to some extent in order to decide on the correct enzyme to cut the sequence at an appropriate place and isolate a worthwhile fragment. Thus, genetic engineering can generally only be carried out once much research has already been done on the organism.

Isolating a DNA Sequence

Based on the chosen and necessary genetic sequences, samples of DNA are collected (chemically isolated from blood or culture). The amount of DNA can be amplified by polymerase chain reaction (PCR) to obtain enough DNA to have a measurable amount of the sequence of interest and to reduce the amount of non-specific cuts in the DNA sequence.

The sequence or gene of interest is isolated using restriction enzymes that cut the sequence out from the DNA strands in the sample and creating “sticky ends”. ThinkQuest has diagrams for clarity. If the isolated DNA does not have sticky ends (some restriction enzymes make blunt cuts, and in some instances the sequence requires such an enzyme because there is not another one that recognizes the region), an additional step is added to create sticky ends in an enzymatic reaction or by using PCR primers that create restriction enzyme sites at the end of the sequence of interest. The DNA sequence may also be altered with site-directed mutagenesis to create a variant sequence.

Genetic Cloning

The vector to be used is also opened up with the same enzymes so the isolated sequence ends will match up. The sequence of interest is ligated (named after the enzyme used – ligase) into the vector, a (usually) bacterial plasmid that allows the DNA sequence of interest to be replicated or gene of interest to be expressed, depending on the aim of the genetic engineering project (many vectors are commercially available for labs to purchase). These sequences are inert – they lack the genes necessary to cause disease. The recombinant DNA is replicated, or cloned, either in vitro via PCR techniques or in vivo in culture (bacterial, yeast, or mammalian tissue culture).

For culture, the vector is inserted into cells via a number of methods, including electroporation and heat shock (called transformation). For bacteria culture, the cells are plated and allowed to grow colonies, which only happens if the DNA entered the cell and the cell survived. The vector usually contains selection genes that allow researchers to choose colonies that only contain the full experimental plasmid. The chosen bacterial colonies are then allowed to grow in culture to increase the amount of the DNA (e.g., for gene therapy). For tissue culture, the cells generally produce the protein or transcript of interest (e.g., insulin) after sequence verification of a clonal population.

Use of the Final Engineered Product

The final product may be used in a number of ways (HowStuffWorks has a list of applications and specifics for each). Viruses may be used to transfer engineered DNA to plants and humans in gene therapy. Blastocysts and seeds may be injected with engineered DNA to create transgenic animals or plants, respectively. The cultures may be grown consistently to produce the protein of choice, such as synthetic insulin and vaccine antigens because the clonal population ensures consistency in the final product. The result is simply a purified genetic sequence of choice, which can then be applied in other technologies.