Dna Replication in the Lab

DNA (abbreviation stands for) replication, often through the polymerase chain reaction (PCR) or bacterial culture, is extremely important in laboratories conducting research on a number of diseases.

When isolated from the blood or cells of a living organism, the amount of any particular portion of DNA is extremely low. In order to obtain enough purified sequence, replication is necessary. This is true for sequencing DNA to determine the presence or absence of particular genes and to create more DNA to be used in experiments or treatments. These techniques exist because of the finite availability of DNA and the need to single out particular portions of very large genomes.

DNA replication is a natural process. Eukaryotes, which include humans and other mammals, exist because of DNA. Inside every cell is the machinery, specifically proteins and nucleic acids, to achieve replication. DNA replication is a step before cell division, because every new cell requires an exact copy of the genome.

Humans have 46 chromosomes comprising approximately 3 billion base pairs. In order to isolate a known, for example, 1000 base pair sequence, PCR is used. The portion of interest is replicated and purified to obtain workable and identifiable quantities. This reason for this is as follows.

From a milliliter of human or animal blood, the commercial kits often used by labs; can isolate, at most, 12 micrograms of DNA. Some labs use their own sets of reagents and may have varying yields, but this is a good estimate of what can be expected. To simply isolate the gene of interest would result in picograms or less of DNA (it is a rough conversion because nucleotides are often considered in g/M), an extremely small amount to work with. Some material is lost in the purification process, which is often through gel extraction (by which such a small amount isn’t even visible) in order to sort out the smaller piece from the larger chromosomes.

Another problem of isolating a gene of interest without replication is that the way to remove the gene is by cutting with a restriction endonuclease, an enzyme that recognizes a short base pair sequence and cuts the DNA at that location. Any endonuclease, of which there are more than 200 commercially available, would cut in multiple places on every chromosome, resulting in a large number of fragments, some possibly the same size, which would contaminate the sample.

The solution is to replicate the portion of DNA of interest using specific primers that recognize a roughly 20-30 base pair sequence on both sides of the sequence or gene of interest. PCR uses similar machinery as the cell for replicating (polymerase and dNTPs), but is limited by the specific primers to that portion of the DNA. There are newer variations of the technique that result in much higher yields, but nanogram to microgram quantities of the DNA of interest can be expected.

When a portion of DNA is required in even higher quantities, bacterial culture is often used. This technique uses the DNA replication machinery present in bacterial cells to replicate a larger piece of DNA a plasmid. Bacterial genomes are unlike those of humans and mammals in that they are circular pieces of DNA, called plasmids. Non-infectious strains of common bacteria, such as E. coli, are commercially available as plasmids that have been manipulated to be safe for both use and disposal. They differ in the restriction endonuclease sites available, selection markers, and size, among other things that vary according to the research being conducted. The DNA sequence of interest is inserted into the cut plasmid using chemical reactions with enzymes, such as ligase, and the whole circular piece is inserted into cells for growth. A selection process occurs to pick out those cell colonies that actually contain the circular plasmid containing the DNA sequence, and then the cells are grown in bacterial medium for a day or however long is optimum for obtaining replication.

The plasmid is then isolated from the cell growth and purified for use in further experiments or the development of therapeutic reagents that require DNA.

These are but two methods that rely on DNA replication in the lab. They are not simple procedures, but have benefited from advances made in both academia and industry. The availability of enzymes and plasmids has been increasing in recent years, making it easier to use these techniques on even more genes of interest. This is important as more and more portions of the human genome are found to be influential in disease, allowing researchers to better understand the role of genetics and to find targets for treatment and prevention.