DNA profiles generated from minute amounts of single cells
Abstract: The genetic code in our cells is built up by deoxyribonucleic acid (DNA) with a sequence that is individual and unique to each person. A cell’s origin can be decided by comparing an established DNA profile with a known profile. The most publicly known application is in the forensic field and its use for identification and for establishing a connection between perpetrators and victims or crime scenes. DNA profiling is also commonly used for kinship investigations. The information embedded in the DNA is also used for diagnostic purposes in conventional medicine. Generating DNA profiles is a well-established procedure, which is used daily and for many purposes. An amount of approximately 150-1500 cells is required to be able to establish a full DNA profile using current methods. There are several situations where the amount of material is limited. To enable analysis where the testing material is limited it is of great value to develop a method that can perform these analyses on minute amounts of cells. If there were a method for generating DNA profiles from single cells then mixed samples from crime scenes would be separable. In tumour biology it is also of interest to obtain information from single cells. The aim with the thesis was to establish the smallest amount of cells needed for a full DNA profile. The thesis started with analyses on extracted DNA. During several experiments dilution series were made to investigate the possibilities to establish profiles from minute amounts of extracted DNA. The main methods used during this thesis were polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE). These methods are well-established tools both in biomedical science and at The Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine. Different factors were optimized and the acquired knowledge resulted in application of DNA on FTA® Micro Cards. The cards are used in the daily routines and are easy to use. Several experiments were then performed on peripheral lymphocytes based on the knowledge acquired during the process. Applying a low amount of lymphocytes on FTA cards proved to be very successful and the method generates DNA profiles at a single cell level. The method is applicable for approximately 5-10 cells.
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