Isolation and Characterization of Buccal Cell Dna Obtained from Mouthwash Samples of Healthy, Tobacco Users and Cancer Patients

The basic sequence of the human genome has been completed and in the coming years a majority of human genes will be identified. The next step is to elucidate the differences among people in sequences, genes, and gene expression patterns to explain what role these differences play in disease, and in some cases to develop genetic tests for these variants. We found that total DNA yield was moderate and enough for PCR amplifications and RFLP. Whole blood cells and buccal cells are commonly collected to obtain a DNA sample. Processing of buccal cells from mouthwash samples is an attractive, non-invasive method for obtaining relatively large amounts of DNA. Once DNA is collected, the genomic DNA must be isolated from other cellular material. Next, a specific region or unknown region must be identified or amplified, performed via PCR (RAPD). Gel electrophoresis is often performed after PCR to verify that PCR was successful and that the amplified target sequence is the correct size. Numerous methods are available to determine a person’s genotype and differ based on allele discrimination and detection. PCR coupled with restriction fragment length polymorphism (RFLP) analysis, a conventional genotyping method, does not rely on automated technology and is practical for laboratories that genotype a limited number of samples. The mouthwash method for obtaining DNA is simple, cost-effective and does not require elaborate instrumentation. In addition, as repeated sampling is possible, there ought to be no paucity for using this DNA for various molecular biology tests, a problem commonly encountered when working with peripheral blood samples. Genomic DNA in mouthwash is stable for prolonged periods at room temperature, and the quantity of DNA recovered from this method is more than sufficient for pharmacogenetic studies. As 4%sucrose, 0.9% saline and branded mouthwashes were used to collect the exfoliated cells, there should be no problem to the donor. The RFLP performed would be useful as a diagnosing tool when gone for Southern hybridization by using a probe. The polymorphism was seen in the gel run and by sequencing the gene which has underwent mutation, can be screened out when sequenced or by using other advanced molecular techniques. In humans, RFLPs were first identified in the vicinity of the globing gene and have been used for diagnosing sickle cell anemia. It can be also used for mapping genes and hence for characterizing genetic defects even if the gene in question is completely unknown. It can be concluded that mouthwash technique for obtaining DNA is a feasible and cost-effective method for large scale epidemiologic studies. The DNA obtained from buccal cells could be utilized for genotyping, DNA finger printing and other genotyping assays. RFLP study based upon comparison of band profile generated after cutting the DNA with Restriction enzymes is a useful tool in identifying polymorphism in individuals. The study showed difference in DNA fragments of Healthy, Tobacco users and Cancer patient generated after RFLP. RAPD-PCR was successful with IFN primer sequence which was of human origin. Hence it can be concluded that tobacco use in any form is injurious to health and might lead to cancer. Therefore Prevention is better than cure