| Background and PurposeDNA extracted from formalin-fixed, paraffin-embedded tissue has always been one challenge plagued many researchers. Formaldehyde as an important component of the commonly used fixative formalin, although it does not produce physical effects of the nucleic acid degradation, but it can cause extensive crosslinking between the nucleic acid and protein, a reaction will cause difficulties of extract DNA from organization. Not only that, if fixative is not buffered neutral formalin solution, formaldehyde will be oxidized to formic acid, the fixative PH value will be substantially reduced, sometimes appear a pH less than1. This acidic environment will lead to DNA strand breaks. These effects lead to the use of PCR amplification technology dissatisfied with the length and number of DNA fragments, which can seriously affect a large number of molecular biology research. DNA extraction from formalin-fixed paraffin-embedded tissue is not easy, but it can not ignore the wealth of information in these organizations for genetics research. A large number of fixed tissue are stored in hospitals around the world, and the Institute of Pathology, they are often the only clue of genetic research study. It is precisely because of this, more and more scholars began to study method of extracting high quality DNA for such fixed tissue, and a series of related papers are gradually being published. Is not optimistic, although the method mentioned in these articles is on the rise, but very few can really achieve the effect of research needs. First, because the more DNA extraction quality measurement method, can not give a same standard to determine whether the effect of extracting DNA worthy; Secondly, although some methods can ensure that the number of the extracted DNA, but because these tissues DNA with more fixed reasons degraded into smaller DNA fragment, despite the considerable number, can not guarantee the fragment length, we are still unable to recognize that this approach is worth universal. In fact, there are many factors that affect the quality of the DNA in fixed tissue, can be roughly divided into three categories. The first category we call fixed before factors which include the type and size of the organization, before it fixed the level of corruption, and so on; fixed factors, including select which fixative, fixative PH value, temperature, and a fixed length of time, etc.; the level of the third category as a fixed factor, e.g. stored in the fixed length of time and storage temperature, and the like. Faced with so many influencing factors, you want to find an extraction method can be very difficult to deal with all the problems.With the increasing number of formalin-fixed paraffin-embedded tissue to be applied to individual identification and paternity cases, we are also facing a new challenge, because most of these organizations are the tumor tissue and malignant tumor. The mutation of the gene coding or non-coding region in malignant tumor tissues is prevalence. This variant is equally likely to occur in the16STR loci in Forensic detection selected to cause difficulties to our identification work.Short tandem repeat (STR) is widely present in eukaryotic genomes and highly polymorphic. Its smaller core sequence is usually2-6bp. The number of repetitions is usually15-30times, so the smaller fragment length, stable PCR amplification results, coupled with highly polymorphic STR loci in the gene transfer process to follow the Mendelian, these characteristics decided PCR-STR multiplex amplification detection technology to become an important means of Forensic technology indispensable. Although, PCR-STR amplification technology is widely used in the organizational source identified cases, but there is a assumption in this detection technology. The assumption is that any individual organizations STR genotypes are entirely consistent. So when the samples are the tumor tissues, this assumption will no longer exist. Studies have found that in the genome of the tumor tissue, whether it is the coding region or non-coding regions are likely to mutate, and the phenomenon also can be observed in the STR loci within the same. Realize this variation in tumor tissue for loss of heterozygosity (loss of of heterozygosity, LOH) and microsatellite instability (the microsatellitte instability, MSI), these changes might impact PTR-STR composite amplification test, lead to the change of the STR genotyping results.Method1.1water bath heating dewaxing:put tissue sample into a1.5ml EP tube, and the tube is filled with1000ul TES (10mmol/LTris1mmol/LEDTA,0.5%SDS, pH8.5) solution at a temperature near the melting point of the paraffin wax (65℃), water bath30minutes and then to13000rpm centrifuged for1minute, the liquid was discarded; above operation is repeated1to2times, and then dried in air at room temperature.1.2microwave heating dewaxing:a1.5ml EP tube equipped with a tissue slice is added200ul of digestion buffer solution (50mmol/LTris lmmol/LEDTA,0.5%Tween-20, pH8.5), sealed and placed in the microwave oven heating1min (time depending on the paraffin how much may be, but not too long), then13000rpm centrifuge for3minutes, discard the liquid; and so repeated four times, and then dried in air at room temperature.1.3xylene dewaxing:a EP tube equipped with tissue sections is filled with1000ul xylene solution, sealed into the shaker slow sway one hour,3minutes at maximum speed centrifugation and then remove and discard the liquid, repeat the operation once;discard the liquid,1000ul ethanol added to the EP tube equipped with tissue sections sealed into the shaker, slow sway three minutes, then the maximum speed centrifuge for3minutes, discard the liquid, repeat the operation once, discard the liquid, and then open EP tube to dry at room temperature.2DNA extraction methods2.1Chelex-100combined with QIAquick(?)PCR Purification Kit method: adding an appropriate amount of Chelex-100solution and proteinase K to the tissue after the completion of the dewaxing processing,56℃for6hours of incubation, denaturation at98℃for8minutes, after13000rpm centrifugation for90 seconds,mixed the supernatant with the PBI solution in a volume ratio of1:5, and then an appropriate amount of the above mixture was added to the purification column, to13000rpm centrifugation for1minute, discard the lower liquid, this operation is repeated until the mixture is all into account. Add300ul PBI solution to purification column to13000rpm centrifugation for1minute, discard the lower liquid. The purification column was washed once by adding prepared PE washing liquid, washed by centrifugation2times for1minute, then residual liquid was volatilized and dried after the purification column is placed in a new1.5ml microcentrifuge tube, open the lid at room temperature for5minutes. Preheated at65℃EB eluent30ul added directly to the silicon film of the purification column, at65℃were incubated for10minutes, and then was13000rpm centrifuged for2minutes to collect the lower liquid used for the PCR reaction or saved backup.2.2Beads extraction method:the sample after the dewaxing was in a1.5ml centrifuge tubes.100ul Lysates prepared was added to the tube, and close lid of the centrifuge tube, heated of95℃for30minutes. Then from the heater the lysate and the sample was transferred to the centrifugal sleeve and centrifuged for2minutes at maximum speed at room temperature, remove the centrifugal sleeve. The vortexing resin storage bottle, so that the resin was suspended, was added to the solution of DNA7UL resin pipette while maintaining the resin was completely suspended. Transient the shock sample/Lysates/resin mixture was incubated at room temperature for5minutes. The tubes on a magnetic rack, to be bead and separated from the solution, the solution was carefully removed, do not touch the wall of the resin. Add100ul lysate, remove the tube from the magnetic rack, a short vortex made the bead suspension, the tube back into the magnetic rack and remove all lysates. Add100ul prepared washing solution, to remove the tube from the magnetic rack with briefly vortexing, the tube was then put back into the magnetic rack, and remove all the washing liquid. The washing of washing liquid was repeated three times, and to ensure that the last time to remove all solution. The tube on magnetic rack was opened the lid, and dried for5minutes in air. Add50ul eluent, close the lid. Brief vortexing made the resin suspension, and heated at65℃water bath for5minutes. Removed and shaked briefly the tube from the water bath, and immediately placed in the magnetic rack. Carefully transfer the solution to the specified tube, standby or storage.2.3Chelex-100combined with DNA IQTM kit method:appropriate amount of the Chelex-100solution and proteinase K added to tissue after the completion of the dewaxing,56℃for6hours of incubation, denatured at98℃for8minutes, and then the purification method of Reference2.2.3. PCR multiplex amplificationTo adopt sinofiler Amplification Kit (Applied Biosystems, United States), and the9700thermal cycler for PCR amplification.4.3130XL Genetic Analyzer electrophoresisSinofiler Amplification Kit:to each sample hole of a96-hole electrophoresis plate, lOul Hi-Di formamide and0.3ul LIZ-500internal standard,1.0ul PCR amplification product or standard allelic ladder was added. After sample addition, the level of the sample plate was centrifuged, placed in3130x1Genetic Analyzer and electrophoresis software analysis GeneMapper3.2.1.5. PCR quantitativeQuantifilerTMuman fluorescence quantitative PCR kit, using the7500real-time quantitative PCR instrument for quantitative PCR.Result1. Comparison of three extraction methodsDifference of DNA content extracted by three different extraction methods was statistically significant (F=13.139, P<0.001); DNA average amount extractedby Chelex-100combined with QIAquickRPCR Purification Kit method is significantly higher than that by bead extraction, Chelex-100extraction combined with bead purification (2.139±3.820versus0.911±2.283, P=0.011;2.139±3.820versus0.709±2.003, P=0.003); DNA content extracted by bead extraction and Chelex-100extract combined with magnetic bead purification method is no statistically significant difference. And the interaction of three extraction and dewaxing method is without statistical significance (F=2.266, P=0.069).2. Comparison of three dewaxing methodDNA content of the three different dewaxing method has a statistically significant difference (F=5.193, P=0.009); xylene dewaxing methods are superior to the other two methods, pairwise comparison P values less than0.05, water bath heating dewaxing method is also better than the microwave dewaxing method (P=0.008).3. Different fixed timeSample tissues were fixed1day,3days,7days, and extracted DNA content showed a clear downward trend, and the difference was statistically significant (F=7.661, P=0.001); and DNA extraction content differences of three extraction methods were statistically significant (P<0.05). There is not interaction between extraction method and fixed time (F=2.266, P=0.069).4. Comparison of the STR typing detection rate of breast cancer tissue and adjacent tissueIn this study, a successful inspection standard defines STR loci for the detection is successfully analysed12and above, and allele peak heights of>100RFU (relative fluorescence units). Group A and group B samples are used xylene dewaxing, Chelex-100combined with a QIAquick(?)PCR Purification Kit purification method for DNA extraction and STR typing, group A with22samples,20samples successfully genotyped, and group B with22samples were not successful genotyping. Correction paired chi-square test between the two results were statistically analyzed, the chi-square value of18.05, P value of less than0.001, STR typing detection rate of breast cancer tissue adjacent tissues, the difference was significant.5. STR loci variation in colorectal carcinoma tissuesIn this research, we compared43colorectal carcinoma tissues with43para-carcinoma tissues. STR testing is successful in43pairs. There is five cases with STR loci variation. The results of2groups were statistically analyzed by the Likelihood ratio test correction methods, the results showed X2=7.240, P=0.007(two-sided), the difference is significant. Conclusion1. Traditional xylene dewaxing, Chelex-100extraction, then purified by the QIAquick PCR purification kit, the extraction of FFPET and STR test results can be positive. The effect of the water bath and microwave heating dewaxing method still can not replace the conventional xylene dewaxing. Comparison from the DNA content and Operational simplicity, preferably FFPET DNA extraction method still is using Chelex-100extraction combined with QIAquick PCR purification kit.2. With fixed time extend by formaldehyde-mediated DNA damage will become more and more serious, leading to tissue DNA extraction and PCR amplification success rate showed a significant downward trend. Effective control tissue fixed period of time, you can improve the success rate of the organizational DNA extraction and amplification. The effect of DNA extraction from the tissue fixedlday is significantly better than the tissue fixed3days or7days.3. STR typing detection rate of breast cancer tissues compared to adjacent tissues was significantly improved. Organizational structure influence on the results. Tissue with less fat and more tumor cells can the success rate of detection.4. STR detection of43cases of colorectal carcinoma and43cases of para-carcinoma paired organizations. Found five cases of colorectal carcinoma tissue variation in15STR loci. And the variation was distributed in D19S433, vWA, D16S539, D2S1338, D18S51, FGA6locus. The variation type included allele increase in2cases, the new allele in1case, allele completely lost in2cases. This result was statistically analyzed by the likelihood ratio test correction method show that compared with para-carcinoma tissues, the mutation rate of colorectal carcinoma tissue in15STR loci used in forensic examination is significant. Therefore, in actual cases of individual identification, you should try to use the normal tissue for testing. When the conditions are not met, and had to use of tumor tissue as samples, if encountered a locus does not match, we must consider tumor genetic instability factors into account, and carefully published the observed results. |
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