Deoxyribonucleic acid (DNA) is a nucleic acid that holds genetic information specifying the biological development of all life forms by establishing the inherited structure of protein in a cell. It consists of two long chains of nucleotides that coil into a double helix shape, which is similar to the form of a winding staircase. It is able to synthesis RNA (ribonucleic acid) and duplicate itself. Each nucleotide of DNA is made up of three different units: a phosphate group, a sugar molecule (deoxyribose), and one of four nitrogenous bases. The bases are either cytosine (C), thymine (T), adenine (A), or guanine (G).
In Eukaryotic cells, DNA is found in the nucleus, and is locked in “threads” called chromosomes. There are 23 pairs of chromosomes found in humans, creating a total of 46. In the entire human genome, the amount of DNA is 2.8 x 109 base pairs in length. In this lab PCR (Polymerase Chain Reaction) was used for the purpose of amplifying the Alu DNA nucleotide sequence found at the TPA25 (tissue plasminogen activator) locus on chromosome.
The Alu insertion is also found on the ACE, PV92, APO, FXIIIB, D1, A25, and B65 loci, and is only present in primates. When testing for the insertion the genotypic results are either be homozygous positive (+/+, present in both alleles), homozygous negative (-/-, present in neither alleles) or heterozygous (+/-, present in one allele but not in the other). The Alu insertion is 300 base pairs in length and classified as short interspersed elements.
There are between 500 and 2,000 restricted to the human genome. Alu DNA does not code for any protein, and is sometimes referred to as “selfish DNA,” as it has no purpose other than to replicate itself. According to previous research, the frequency of having the Alu insertion is lowest in the African population, at 0.463. India has second lowest at 0.544, the Asian population follows with a frequency of 0.557, and the highest frequency of the Alu insert comes from Europe, at 0.559.
Cheek cells were the source of the template DNA that was isolated. Thousands of cells were collected by using a saline mouthwash and placed in a centrifuge to separate them from other unwanted debris. The cells were then resuspended in a solution containing Chelex beads, which were negatively charged. The Chelex beads play an important part in preventing DNAse from tearing apart the DNA strands. DNA is also negatively charged, and Ca2+ and Mg2+ ions from inside the cell aid DNAse to cut up DNA strands. The Chelex beads allow the Calcium and Magnesium ions to join to them instead of the DNA, effectively preventing DNA from being torn apart by the enzyme DNAse. The cells are then lyzed by boiling to break open the cell and nuclear membranes, exposing the DNA. The DNA is extracted and replication is begun via the Polymerase Chain Reaction.
The two paragraphs prior the asterisks were taken directly from my previous lab, “Extracting and Isolating DNA” Kary Mullis invented the Polymerase Chain Reaction in 1983, and ten years later won the Noble prize for his innovation. Other methods of replicating DNA required a living organism such as yeast or E. Coli, while this technique only calls upon an enzyme and temperature cycling. A very small amount of DNA is able to be amplified into large quantities over a relatively short period of time.
The PCR reaction takes place within a thermal cycler. This machine is able to rapidly and accurately adjust temperature at different parts of the reaction to fully accommodate each step necessary. There are four major components necessary to successfully amplify DNA through PCR; a DNA template containing the DNA to be copied, Taq (Thermus aquaticus) DNA polymerase, primers that establish the beginning and end of the region to be increased, free nucleotides, and buffers within the reaction.
There are three main steps involved with one cycle of PCR. Denaturing is the first step, where the solution the template DNA is in is be heated to 94-96ï¿½C. This breaks the Hydrogen bonds that hold the two strands together, and also activates the Taq DNA polymerase, which is thermostable. Originally DNA polymerase needed to be replenished after every heating cycle because it would be destroyed at the temperature needed for DNA hydrogen bonds to be broken. However thermophilic bacteria were found living around geysers in temperatures that exceed 110ï¿½C. The DNA polymerase extracted from these organisms was not destroyed at the high temperatures needed for PCR, making the process much more efficient. The most common DNA polymerase used in PCR comes from the Bacterium Thermus Aquaticus.
Following denaturing, a process called annealing takes place, in which primers need to attach themselves to the DNA strands to mark the region that will be amplified. Primers are short strands of DNA – usually 18 to 25 base pairs in length – that are complementary to the ends of the DNA fragment to be copied. Annealing takes place at temperatures ranging from 45-60. The temperature entirely depends on the primers used, and it is usually about 5C lower than their melting point. The primers are the point where the DNA polymerase begins to synthesize the new DNA strands. This final step is called extension, and the temperature at which it takes place at is usually around 72C.
The whole cycle of PCR does not take up a lot of time. Denaturing takes between two and five minutes, annealing takes one to two minutes, and extension takes about one minute. However multiple cycles are needed to amplify the DNA to proper amounts – around 30 cycles in total, depending on the efficiency of the process and the size of the template DNA. After DNA has been amplified, it must be subjected to gel electrophoresis.
Gel electrophoresis is the process by which DNA strands are separated on the basis of electric charge, size (base pairs), and shape and then compared to predetermined lengths of DNA. To separate the molecules an electric charge is applied to a gel matrix and since DNA is negatively charged it travels towards the anode. Agarose, a colloid extracted from seaweed is one of the most common materials used to make the gel. The pores in agarose are extremely large, and when compared to polyacrylamide gels the final resolution is inferior. Ethidium bromide is also added to the gel, as it fluoresces under UV light and makes the bands of DNA distinct and visible.