Material and Method: A combination of short tandem repeats (STR) of the human genome consisting of CSF1PO, TH01, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539 and Penta E were selected on the basis of ease of application and bioinformatic discrimination power. Possible forms of diagnostic tissue mix up were set in 3 different models with 3 diagnostic tissue samples of 2 different cases. Of the tissue samples selected, A (salivary gland) and B (striated muscle) belonged to the same case and C (uterus wall) belonged to another case. In the first model, there was no problem about tissue identity (M1: A/B). In the second model, two different diagnostic material were mixed up (M2: B/C). In the last model, there were 3 diagnostic material obtained from 2 different cases (M3: A/B/C). DNA was extracted from all tissue samples and all of the selected 10 STR were amplified with specially designed primers by PCR. After chemical denaturation, amplicons were submitted to polyacrylamide gel electrophoresis for discrimination of single DNA strands according to their conformation polymorphism (SSCP). Special patterns of each STR in the gel matrix obtained from M1, M2 and M3 models, were evaluated on the principle of being ‘same or different' to determine the diagnostic material identity.
Results: Each of the salivary gland, striated muscle and uterus wall samples were correctly identified (matched with the right source cases) after evaluating 10 different STR SSCP patterns designed under M1, M2 and M3 models.
Conclusion: This application targeting to solve diagnostic tissue identity problems is a simple and cheap application of SSCP and its efficacy was proven on the designed models.
We define a procedure aiming to remove any suspicion of specimen mix-up and to determine sample ‘ownership' in this study. A unique feature of the human genome is used for this procedure. Although the pattern of the four letters that form the sequence for the human genome are generally the same (>99.9%) the remaining part has regions that make every individual unique. One of these special human genome regions have been defined as short tandem repeats (Short Tandem Repeats: STR)[1-4]. The human genome is generally a book that can be written with the four letters of A (adenine), T (thymine), G (guanine) and C (cytosine) and has many repeated letter sequences. Those repeats where the number of repeated letters are 2 to 6 are named ‘short'. The letters of this short sequence and the number of consecutive repeats, i.e. the ‘sequence and number of repeats' (STR) is different for each genome[1-4]. The features of the STR regions defined in the human genome are an open source accessible to everyone[5]. As can be seen in this source, there are many STR regions that have been defined in the human genome. However, some of these are only useful to ‘differentiate the genome'. It is possible to greatly increase our discrimination ability by increasing the number of STR's with selections from this region. STR combinations with a very high discrimination values can be found using bioinformatic analysis of data obtained from data banks. However, professional studies using the earth's population and their geographic characteristics together with some other technical details provide the logical STR combinations in data banks.
We defined an easy-to-use and low cost molecular technique to determine “ownership of the sample” and solve possible diagnostic material mix-ups in pathology practice by creating various experimental models in this study. We created a combination based on bioinformatics that included 10 separate STR regions to create a differentiation power that was valid but did not have to be very sensitive. The easy use of the PCR technique to be used for STR analysis and the differentiating power of the polyacrylamide gel electrophoresis to be performed afterwards were taken into account in creating this combination.
Possible diagnostic material mix-up was modelled in 3 ways taking the practice of pathology into account. Model 1: There is no diagnostic material mix-up in this model. Salivary gland (A) and striated muscle tissue (B) from the same person were used. Model 2: The material from two different patients was mixed up. Striated muscle (B) and uterine wall (C) samples from two different persons were used. Model 3: There was mix-up between three pieces of diagnostic material from two different patients. Salivary gland (A) and striated muscle tissue (B) from one person and uterine wall (C) from another person was used.
We made it possible to use an important control parameter (gold standard) for ‘ownership' by selecting tissues that can be easily differentiated microscopically.
We performed DNA extraction (QIAamp DNA mini kit, QIAGEN, Hiden, Germany) after obtaining 4 sections 10 μm thick from the paraffin blocks of the selected tissue samples. The 10 selected STR regions were amplified using a PCR reaction mixture and thermal cycle with the obtained DNA as template (Table I). The amplicons were observed in 2% agarose gel and cleaned with the spin column technique. Each STR region that was cleaned and purified was converted to single strand form by chemical denaturation. Electrophoresis for 2 hours at 80 volt constant current in a 7% polyacrylamide gel matrix was used for differentiation on the basis of the nucleic acid sequence specific conformation of each strand (Single Strand Conformation Polymorphism: SSCP). The gel was evaluated in a UV imaging system after staining with ethidium bromide. The ownership of the diagnostic material in the M1, M2 and M3 models were evaluated using the ‘same or different' principle for the specific conformation patterns created as separate columns for each STR region in the gel matrix. The same pattern in the compared STR regions showed that the two materials were from the same person while a different pattern in at least one STR region indicated that the samples were from different persons.
In conclusion, this procedure that aims to eliminate diagnostic sample mix-up is a simple and inexpensive SSCP application and mix-up models have demonstrated its effectiveness. The rationale may seem complicated but pathologists will find it easy to understand and it is simple enough to be used in any laboratory with the technical capacity to perform PCR and electrophoresis.
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