Single Nucleotide Polymorphisms as Human Disease Markers Essay Example for Free

Single Nucleotide Polymorphisms as Human Disease Markers Essay In using single nucleotide polymorphisms (SNPs) to match subtle DNA changes with an individual’s susceptibility to a disease of interest, one can start by distinguishing the suspect DNA from a preset standard using the technique of restriction fragment length polymorphism (RFLP). In this method, copies of extracted and purified DNA are made using the polymerase chain reaction (PCR) before being cut up by preselected restriction endonucleases, and the resulting fragments subjected to electrophoresis. Once a suspect sequence in the fragments obtained is found, it can then be subjected to a sequencing technique that can pinpoint the nature of the SNP. One such method is Pyrosequencing, where a sequencing primer is prepended to the fragment under scrutiny and is mixed with a cocktail composed of a DNA polymerase, ATP sulfurylase, luciferase (the enzyme responsible for making luciferin in luminous animals glow), and apyrase, as well as luciferin and adenosine 5’-phosphosulfate (APS). Deoxyribonucleotide triphosphates (dNTPs, four in total, corresponding to the four DNA bases) are added, one by one, into the reaction mix. For each time a dNTP is added, the polymerase causes the incorporation of the dNTP into the strand at appropriate positions, if any are present, with the simultaneous release of pyrophosphate, in an amount that is equimolar to how much nucleotide was used in incorporation. The pyrophosphate reacts with the APS, catalyzed by ATP sulfurylase, to produce ATP which participates in the chemoluminescence reaction of luciferin and luciferase. The amount of visible light generated by the reaction is proportional to the amount of ATP generated, and this is recorded by a special camera and noted as a peak into what is called a Pyrogram. How much light is generated depends on how much dNTP was used up. Any unused dNTP and ATP present in the mix is degraded by the apyrase. The sequence of the fragment can then be read off from the peaks of the gradually being produced Pyrogram. Specially constructed software can then spot SNPs by comparing the Pyrogram of the fragment of interest with known Pyrograms. By using an â€Å"association study†, where people with the trait and a control group are compared with regards to the presence of â€Å"marker alleles†, or alleles that can be shown to be present only in people with the disease. SNPs are convenient for this purpose since SNPs are easily inheritable and occur usually more frequently in genes than the other possible variations. This is most useful for diseases that can be shown to be influenced by only a single gene, for association is difficult enough when done on a single gene. Accuracy in identification can be had by having the affected and the control be as closely related as possible, since differences that might be disease-related will be easier to spot. References: Ronagi, M. Elahi, E. (2002). Discovery of single nucleotide polymorphisms and mutations by Pyrosequencing. Comparative and Functional Genomics, 3(1), 51-56. Li, M., Boehnke, M., Abecasis, G. R. (2006). Efficient study designs for test of genetic association using sibship data and unrelated cases and controls, The American Journal of Human Genetics, 78, 778–792.