Difficult Degradation Of Dna Testing Of Samples

Objective: To take degraded DNAremains in the aged bones,teeth,formalin fixed and paraff in embedded tissues (FFPET) as the research subject, and adopts and compares different techniques and methods to find a suitable method for degraded sample extraction. In addition, methods for extracting other common micro degraded samples will be established and the application value of MiniFiler kit in STR profile of degraded samples will be appraised.Method: 10 samples with test resμlts have been selected, and decalcification effect, digestive ability of different lysis buffer and different purification methods of each sample have been compared respectively. An effective extraction method for OEB lysis buffer organically extracted to associat with QIA quick Spin Column has been established at this basis, and this method has been applied to 24 bone samples. We selected 10 samples of FFPET from the hospitals; took the samples after different pretreatment for mμltiplex PCR tests to compare the influence of different pretreatment measures on DNA extraction; selected a group of samples and extracted DNA from the two samples with the organic method and Chelex-100 method respectively and compared the results generated by the two methods as well as the DNA of the two samples; observed the response of genetic mutation of cancer tissues on PCR and influences on the result analysis.Compared the testing resμlts of MiniFiler? and IdentiFiler? kits in 13 general quantity biological samples , including blood stains, sperm stains,tissues, bones and so on. Meanwhile the sensitivity of these two kits were also compared.Result: It's proved that the results with pre-decalcification are obviously better than those without decalcification; there is no significant difference of digestive abilities between OEB and silica-based method lysis buffer; the test of using QIA quick Spin Column has obvious advantage over Microncon 100. All of the 23 bone samples applying the new method have been examined by over 12 STR Locus, from the 23 bones and teeth samples, we acquired over 12 STR genetic locus results; We took 10 samples, the profile patterns of the sample groups that was dewaxed for 2 times for 2 and 12 hours at room temperature have no difference from that of the l0min group dewaxed at 37°C, while the peak value in profile pattern of the sample dewaxed 2 times at 37°C are better than the results above2, which means that the samples dewaxed 2 times at 37°C are dewaxed more thoroughly. After QIA quick purifiers are extracted with the organic method, 9 of the 10 samples got complete STR results and only 1 sample got merely part of the genetic locus profile result. Using Chelex-100 method, none of the 10 samples got STR profile results. We also concluded that the genetic mutation of cancer tissue will alter the result of STR profile, since we found recombination, loss of alleles, and unbalanced peak value in the 10 paraffin section samples of cancer tissue; compared the testing results of MiniFiler kit and IdentiFiler? kit in 13 samples.Conclusion: With its stability and efficiency this method is applicable to the DNA test of samples. Properly adopting these methods—raising temperature, prolonging the time of dewaxing, or combining organic extraction with QIA quick purifier method—can all improve the success rate of DNA test. The results demonstrated that the MiniFiler? kit can markedly improve the typing success rate of degraded biological samples, and is suitable for analyzing difficult biological samples in forensic practice.