| Formalin-fixed and paraffin-embedded tissues, due to their ability to preserve organizations for longer time or the requirements of the preparation of test specimens, and therefore they are often used in clinical pathology, forensic pathology and medicine scientific research. Although this process can prevent DNA from the damage of endogenous nuclease, the formaldehyde will not only lead to the break and degradation of the DNA strands, depurination, cross-linking between DNA and protein, resulting in the change of the quality of DNA in fixed tissues, but also inhibit PCR amplification when its residual stay in the template DNA.Studies had shown that many factors can affect the DNA which was extracted from formalin-fixed and paraffin-embedded tissues, such as the type of fixative, the fixed-time, the thickness of the slice, the storage-time of the slice and the different treatment methods. By optimizing the conditions of protease digestion, increasing cycle times or using nested PCR and so on, even though these could increase the amount of PCR products or specificity, these did not significantly improve the success rate. By shortening the length of the fragments, such as the development of miniSTR system or SNP system, although enhancing the success rate of the sub-type of the degrade DNA Samples, there were still many problems, such as the loss of alleles or loci, unbalanced peak, pseudo-peak and so on. In addition, the length of amplified fragment in miniSTR system could not be reduced infinitely (50～200bp). If SNP analysis technology wanted to achieve the same recognition rate with the using STR kits, it required more loci, this restricted its applications in real cases.This study was based on the needs of forensic science, combined with the results of previous studies, by improving the ammonium acetate extraction method, and combined with pre-PCR repair technology, in order to provide a new approach for DNA-STR typing of formalin-fixed and paraffin-embedded tissues in forensic cases.Material and Methods1. The kidney tissues are obtained from the body in the death of 24 hours, their sizes were 1cm×1cm×0.5cm, together with 15ml of the heart-blood were provided by the Faculty of Forensic Medicine in China Medical University.2. Paraffin embedded after fixing with 10%neutral formalin solution, the fixing time was 1d,3d,5d and 7d, then slicing for the experiment.3. Extracted DNA from the blood, the fresh kidney tissues and the FFPET, and then analyzed the influencing factors of the ammonium acetate extraction method, then compared it with phenol/chloroform extraction method, Chelex-100 extraction method, and we also studied the smallest FFPET samples which could achieve successful classification in forensic science.4. Repaired the template DNA using repair enzymes, which extracted with improved Ammonium acetate extraction method.Result1. PCR-PAGE of the genomic DNA which extracted with different dewaxing methods showed that non-dewaxing and dewaxing all can get clear bands; but for the intensity, the water bath of 95℃get the brightest bands, followed by 65℃, xylene, microwave and non-dewaxing.2. PCR-PAGE of the genomic DNA extracted with different digestion buffer showed that digesting with 0.5%Tween 20 could get the brightest bands, followed by digesting with 1%Triton X-100,0.5%SDS and 2%CTAB.3. From the agarose gel electrophoresis of the genomic DNA, which extracted by different concentrations of ammonium acetate solution, we could see that there were no significant differences between the amounts of the DNA. For the PCR-PAGE, it showed that they all could get clear bands, and there were no significant differences of the brightness between them.4. The agarose gel electrophoresis of the genomic DNA which extracted from different fixed-time showed that the electrophoretic bands were all dispersed into shape, and with the extension of the fixed-time, the bands dispersed gradually, and the brightest regions gradually moved down. PCR-PAGE showed that DNA extracted from different fixed-time all can get clear bands, but with the extension of the fixed-time, the bands gradually weakened.5. The agarose gel electrophoresis of the genomic DNA which extracted from different methods show that the bands from Chelex-100 extraction method are brightest, followed by from the improved ammonium acetate extraction method and from the phenol/chloroform extraction method; The internal comparing of one method showed that with the reduction of the amount of the samples, the electrophoretic bands all weakened in the three methods of genomic DNA extracted. OD260/OD280 showed that it was between 1.6 and 2.0(1.962±0.195) of the genomic DNA extracted by the improved ammonium acetate extraction method, by the phenol/chloroform method it exceeded 2.0 (2.110±0.470), by the Chelex-100 method it was about 1.0 (1.018±0.124). From the amount we could see that, The Chelex-100 extraction method get the maximum of the genomic DNA, followed by the improved ammonium acetate extraction method and the phenol/chloroform extraction method, and it also showed that the fewer the material, the bigger the trend was. From the PCR-PAGE, the results told us that, we could get clear bands by the improved ammonium acetate extraction method; at the same time they were weaker by the phenol/chloroform extraction method, and the fewer the material, the weaker the bands were, when the samples was reduced to 1/8, we nearly get no bands; When the samples were 1 piece, the genomic DNA extracted by the Chelex-100 extraction method can not get clear bands, when they reduced to 1/2, the bands were undefined, while when they reduced to 1/4 and 1/8, we could get clear bands, but they were still weaker than the bands from the improved ammonium acetate extraction method.6. Pre-PCR repair of the template DNA which extracted by the improved ammonium acetate extraction method show that, there were stronger bands in repair group with Taq DNA polymerase, while the control group had no band or weaker bands; There were no significant differences between the repair group and the control group when using T4 ligase for repair; when we used Taq DNA polymerase joining with T4 ligase for repair, we get clear bands at last.Conclusion1. The improved ammonium acetate extraction method has good qualities of simple, economical, non-toxic and so on; when it compared with phenol/chloroform extraction method and Chelex-100 extraction method, its DNA extracted get higher purity and amount; it is suitable for micro-samples.2. The DNA repair techniques with Taq DNA polymerase can greatly enhance the success rate of STR genotyping for high degrade samples, which is suitable for the STR genotyping of high degrade samples.3. Using improved ammonium acetate extraction method joining with DNA repair technique can improve the success rate of STR genotyping for the minimum FFPET Samples (0.5cm x 0.25cm x 7μm) in forensic science, which has an important significance for personal identification of formalin-fixed and paraffin-embedded tissues.
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