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Introduction
The purpose of this project is to find a way to test for the presence of an allele which serves as a marker for predisposition to develop Post Traumatic Stress Disorder (PTSD). This allele is present in many adults, some are homozygous for the trait and others are heterozygous. They will be herein referred to as the Long or Short allele. Studies have correlated the presence of the short allele with a predisposition to develop PTSD. Since PTSD is a complex human behavioral disorder, of which the entire etiology is not known, the presence of this allele represents only a small piece of information in a highly complex web of neurobiological function.
The development of this test is crucial in preventing onset of the disease by reducing exposure to traumatic events like combat for example. Genetic testing is expensive and a more cost effective way to detect vulnerability is needed. A test that requires minimal training to perform and with materials that are cost-effective. So far, we have been able to repeat previous experiments to amplify by PCR the sequence of variance (Mellman et al. 1996, Gerlerntner et al. 1993). This has been done by taking blood samples from donor volunteers. We used the GC rich from Roche along with the Taq PCR all in one tube and imaged using etBR (ah! I'll put the detail of kits in the final paper) We can use PCR and gel electrophoresis as a way to test our results we get with the new method. What is the most cost effective, simple way to test for allelic variation?
In order to improve upon previous work, we plan to use a less invasive approach to scan DNA samples: Urinalysis. There is a kit from Qiagen which claims to extract an average 25ug of DNA from urine samples. We can first utilize this kit and later develop our own protocol to quickly extract DNA from urine samples. Due to the 44bp deletion in the short version of the allele, we will use a generated probe that will hybridize to the region of interest located in the long allele. The probe should be designed in a way that it can recognize the sequence, incubation/hybridization period is short and visualization of the results can be seen without the use of special equipment or machines. The probe hybridization presents the biggest challenge in this project. Is there a way to develop the probe so that it can hybridize quickly and consistently? Southern blotting by way of random-oligo-primed-synthesis is a widespread method used to probe for specific sequences. Although this is typically used in conjunction with Polymerase Chain Reaction using expensive enzymes.
Is there a way to bypass this part of the process? Can we utilize other ideas in molecular biology to solve this problem? Often, people use immunohistochemistry (want to expand on this idea) to detect the presence of certain compounds expressed in the cell. This is the case in Western Blotting but often we are detecting using antibodies, but can a similar approach be used for our purpose? Approaches using liposomes to detect hybridization have been used frequently in immunology studies, perhaps there is a way to expand on those ideas. In designing the probe, I would like to combine a variety of approaches to be able to detect hybridization of the probe and then visualize the results. Paper chromatography, as used in the fields of chemistry as well as molecular biology is useful for quick visualization to detect the presence of a molecule/compound using reactive dyes that simply use nitrocellulose paper along with chemical tagging to produce a visual reaction (Zhang et al. 2000, Rule et al. 1996). Using a known genotype positive for the long allele as a control, we should conduct testing simultaneously using the probe and paper chromatograph. We can quantify our results by using a method similar to statistical analysis of Western Blotting. If the allele is present, there should be two positive reactions indicated by color change and if the allele is not present, one positive (control) and one negative reaction.
Budget
In development
Bibliography/References
Rule et Al. 1996
Gerlerntner 1993
Mellman 1996
Zhang et Al. 2000